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ABSTRACT: The adaptive response to hydrogen peroxide (H(2)O(2)) in Pseudomonas aeruginosa involves the major catalase, KatA, and OxyR. However, neither the molecular basis nor the relationship between the aforementioned proteins has been established. Here, we demonstrate that the transcriptional activation of the katA promoter (katAp) in response to H(2)O(2) was abrogated in the P. aeruginosa PA14 oxyR null mutant. Promoter deletion analyses revealed that H(2)O(2)-mediated induction was dependent on a region of DNA -76 to -36 upstream of the H(2)O(2)-responsive transcriptional start site. This region harbored the potential operator sites (OxyR-responsive element [ORE]) of the Escherichia coli OxyR binding consensus. Deletion of the entire ORE not only abolished H(2)O(2)-mediated induction but also elevated the basal transcription, suggesting the involvement of OxyR and the ORE in both transcriptional activation and repression. OxyR bound to the ORE both in vivo and in vitro, demonstrating that OxyR directly regulates the katAp. Three distinct mobility species of oxidized OxyR were observed in response to 1 mM H(2)O(2), as assessed by free thiol trapping using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. These oxidized species were not observed for the double mutants with mutations in the conserved cysteine (Cys) residues (C199 and C208). The uninduced transcription of katAp was elevated in an oxyR mutant with a mutation of Cys to serine at 199 (C199S) and even higher in the oxyR mutant with a mutation of Cys to alanine at 199 (C199A) but not in oxyR mutants with mutations in C208 (C208S and C208A). In both the C199S and the C208S mutant, however, katAp transcription was still induced by H(2)O(2) treatment, unlike in the oxyR null mutant and the C199A mutant. The double mutants with mutations in both Cys residues (C199S C208S and C199A C208S) did not differ from the C199A mutant. Taken together, our results suggest that P. aeruginosa OxyR is a bona fide transcriptional regulator of the katA gene, sensing H(2)O(2) based on the conserved Cys residues, involving more than one oxidation as well as activation state in vivo.
Journal of bacteriology 11/2009; 192(2):381-90. · 3.94 Impact Factor
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ABSTRACT: Phage therapy against Pseudomonas aeruginosa infections has received renewed attention owing to the increasing prevalence of antibiotic resistance in this bacterium. Here, we isolated and characterized two new potentially lytic bacteriophages (MPK1 and MPK6), which produced large and clear plaques on P. aeruginosa strain PAO1. Based on their morphology, MPK1 belongs to the Myoviridae, while MPK6 belongs to the Podoviridae. The group B polysaccharide of lipopolysaccharide was required for infection, suggesting that their host spectra are associated with the serotypes of P. aeruginosa strains. Intramuscular and intraperitoneal administration of MPK1 and, to a lesser extent, MPK6 significantly protected mice from mortality caused by PAO1-induced peritonitis-sepsis (P < 0.01). Mice treated with either phage also had lower bacterial burdens in their livers, lungs, and spleens. The antibacterial efficacy of MPK1 and MPK6 was also evaluated based on Drosophila melanogaster systemic infection caused by P. aeruginosa, for which phages were administered by feeding. Both phages significantly delayed the PAO1-induced killing of D. melanogaster (P < 0.001), although MPK1 persisted longer than MPK6 in uninfected D. melanogaster tissue samples. These results suggest that a mini-scale experiment using D. melanogaster infection is valid for evaluating the antibacterial efficacy of phage therapy against P. aeruginosa infections.
Antimicrobial Agents and Chemotherapy 04/2009; 53(6):2469-74. · 4.84 Impact Factor
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ABSTRACT: Pseudomonas aeruginosa is an opportunistic human pathogen that produces and secretes exopolysaccharides (EPS), in which cells are embedded to form a highly organized community structure called biofilm. Here, we characterized the role of cyclic diguanylate (c-di-GMP) and EPS (PEL) overproduction in the wspF mutant phenotypes of P. aeruginosa PA14 (wrinkly appearance, hyperadherence, impaired motilities, and reduced virulence in acute infections). We confirmed that the elevated c-di-GMP level plays a key role in all the wspF mutant phenotypes listed above, as assessed by ectopic expression of a c-di-GMP-degrading phophodiesterase (PvrR) in the wspF mutant. In contrast, PEL EPS, which is overproduced in the wspF mutant, was necessary for wrinkly appearance and hyperadherence, but not for the impaired flagellar motilities and the attenuated virulence of the wspF mutant. These results suggest that cdi- GMP affects flagellar motility and virulence, independently of EPS production and surface adherence of this bacterium.
Journal of Microbiology and Biotechnology 08/2008; 18(7):1227-34. · 1.38 Impact Factor
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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.
Infection and immunity 07/2008; 76(9):4152-62. · 4.21 Impact Factor
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ABSTRACT: A temperate transposable bacteriophage (MP22) was isolated from a Korean clinical isolate of Pseudomonas aeruginosa. It has a coliphage lambda-like morphology and a double-stranded DNA genome. The complete nucleotide sequence and annotation of the MP22 genome and its characteristics are presented. The MP22 genome is 36 409 bp long with a G+C content of 64.2 mol%. The genome contains 51 proposed ORFs, of which 48 (94 %) display synteny and significant nucleotide and protein sequence similarity to the corresponding ORFs of the closely related phage, D3112. Three of the predicted ORFs are unique proteins, whose functions are yet to be revealed. The phage c repressors exhibit striking dissimilarities and, when present as a single gene, did not show cross-immunity. In contrast, although an MP22 lysogen could be productively infected with D3112, MP22 could not grow on a D3112 lysogen, indicating a role of other D3112 genes in superinfection exclusion.
Microbiology 10/2007; 153(Pt 9):2885-95. · 3.06 Impact Factor
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ABSTRACT: An opportunistic human pathogen, Pseudomonas aeruginosa, contains the major catalase KatA, which is required to cope with oxidative and osmotic stresses. As an attempt to uncover the H2O2-dependent regulatory mechanism delineating katA gene expression, four prototrophic H2O2-sensitive mutants were isolated from about 1,500 TnphoA mutant clones of P. aeruginosa strain PA14. Arbitrary PCR and direct cloning of the transposon insertion sites revealed that one insertion is located within the katA coding region and two are within the coding region of oxyR, which is responsible for transcriptional activation of several antioxidant enzyme genes in response to oxidative challenges. The fourth insertion was within PA3815 (IscR), which encodes a homolog of the Escherichia coli iron-sulfur assembly regulator, IscR. The levels of catalase and SOD activities were significantly reduced in the iscR mutant, but not in the oxyR mutant, during the normal planktonic culture conditions. These results suggest that both IscR and OxyR are required for the optimal resistance to H2O2, which involves the expression of multiple antioxidant enzymes including KatA.
Journal of Microbiology and Biotechnology 09/2007; 17(8):1344-52. · 1.38 Impact Factor
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ABSTRACT: R-type pyocin is a bacteriophage tail-shaped bacteriocin produced by Pseudomonas aeruginosa, but its physiological roles are relatively unknown. Here we describe a role of R-type pyocin in the competitive growth advantages between P. aeruginosa strains. Partial purification and gene disruption revealed that the major killing activity from the culture supernatant of PA14 is attributed to R-type pyocin, neither F-type nor S-type pyocins. These findings may provide insight into the forces governing P. aeruginosa population dynamics to promote and maintain its biodiversity.
Journal of Microbiology and Biotechnology 02/2007; 17(1):180-5. · 1.38 Impact Factor
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ABSTRACT: Infection of Drosophila melanogaster adults with 6 Vibrio species revealed that V. cholerae was lethal (100% mortality) within 20 h as a result of systemic infection. Avirulent infection by V. vulnificus restricted the subsequent virulent infection by V. cholerae. The immediate transcription of antimicrobial peptides (AMPs), most notably Attacin A, was delayed in V. cholerae infection compared to V. vulnificus infection. Ectopic expression of Attacin A and Metchnikowin enhanced the survival of D. melanogaster upon V. cholerae infection. These results suggest that AMPs are important in the response to infections by Vibrio species and that the signaling pathways governing their expression may be targeted by V. cholerae virulence factors to elude the innate immunity of Drosophila.
Molecules and Cells 01/2006; 20(3):409-15. · 2.18 Impact Factor
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ABSTRACT: The multi-host pathogen, Pseudomonas aeruginosa, possesses an extraordinary versatility which makes it capable of surviving the adverse conditions provided by environmental, host, and, presumably, competing microbial factors in its natural habitats. Here, we investigated the P. aeruginosa-Bacillus subtilis interaction in laboratory conditions and found that some P. aeruginosa strains can outcompete B. subtilis in mixed planktonic cultures. This is accompanied by the loss of B. subtilis viability. The bactericidal activity of P. aeruginosa is measured on B. subtilis plate cultures. The bactericidal activity is attenuated in pqsA, mvfR, lasR, pilB, gacA, dsbA, rpoS, and phnAB mutants. These results suggest that P. aeruginosa utilizes a subset of conserved virulence pathways in order to survive the conditions provided by its bacterial neighbors.
The Journal of Microbiology 11/2005; 43(5):443-50. · 1.10 Impact Factor
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ABSTRACT: We demonstrate that among the three monofunctional catalases of Pseudomonas aeruginosa PA14, KatA and, to a lesser extent, KatB, but not KatE, are required for resistance to peroxide and osmotic stresses. KatA is crucial for adaptation to H2O2 stress and full virulence in both Drosophila melanogaster and mice. This dismantling of catalase roles represents a specialized catalytic system primarily involving KatA in responses to adverse environmental conditions.
Infection and Immunity 08/2005; 73(7):4399-403. · 4.16 Impact Factor
