Pseudomonas aeruginosa OxyR Is Required for Full Virulence in Rodent and Insect Models of Infection and for Resistance to Human Neutrophils
http://www.100md.com
感染与免疫杂志 2005年第4期
Departments of Internal Medicine
Molecular Genetics, Biochemistry and Microbiology
Division of Pulmonary and Critical Care Medicine, University of Cincinnati College of Medicine
Cincinnati VA Medical Center, Cincinnati, Ohio
TEXT
The role of the H2O2-responsive transactivator OxyR was evaluated in mouse and Drosophila models of Pseudomonas aeruginosa infection and by assays of neutrophil killing. Relative to that of wild-type bacteria, oxyR mutant viability was reduced by 99% or greater in mouse models of acute pneumonia and burn sepsis, and the oxyR mutant was more sensitive to killing by human neutrophils and was delayed in the kinetics of Drosophila melanogaster killing.
During human infection by the opportunistic bacterium Pseudomonas aeruginosa, the organism faces exposure to toxic reactive oxygen intermediates generated by phagocytic cells. During the phagocytic oxidative burst, hydrogen peroxide (H2O2) is generated at high millimolar levels within the phagosomal vacuole (4). Enzymatic defenses against H2O2 in P. aeruginosa are provided by at least three catalases (KatA, KatB, and KatC) (2, 8), several probable peroxidases (refer to www.pseudomonas.com), and four established alkyl hydroperoxide reductases (AhpA, AhpB, AhpCF, and Ohr); members of the last class can also degrade H2O2 and various alkylhydroperoxides (9). The major gene product involved in endogenous H2O2 detoxification in P. aeruginosa is the 170-kDa heterotrimeric catalase KatA (8). Accordingly, katA gene expression is consistently high during vigorous aerobic growth. In fact, KatA activity is maintained at such high levels that even significant H2O2 stress triggers only a twofold increase in expression, suggesting that a high rate of KatA activity is critical for the detoxification of endogenous H2O2 produced during normal aerobic metabolism. In contrast to that of the katA gene, expression of several other oxidative stress defense genes, including katB-ankB (5), ahpB, and ahpCF, is dramatically increased upon exposure to H2O2, organic peroxides, or the redox cycling agent paraquat, suggesting that a highly specific and tightly regulated stress response exists in this organism (5, 9). We have found that such a response is governed by the 34-kDa H2O2-responsive transactivator known as OxyR in P. aeruginosa (9). P. aeruginosa lacking OxyR is exquisitely susceptible to H2O2, even though it possesses wild-type catalase activity (9). In fact, isolated colonies of oxyR mutant bacteria do not even appear on aerobic Luria-Bertani (LB) agar, because autoxidizable components in the medium itself generate 1.2 μM H2O2/min (3). This concentration of H2O2 has been detected in peripheral blood from human donors and is sufficient to kill these organisms (3).
In this study, we tested the hypothesis that OxyR is required for the full virulence of P. aeruginosa by use of (i) a mouse intranasal model of acute pneumonia, (ii) a mouse burn sepsis model, (iii) an in vitro model of neutrophil killing, and (iv) a Drosophila melanogaster alternative model for animal infection.
The P. aeruginosa oxyR mutant is impaired in causing pneumonia and infecting burn wounds in mice. P. aeruginosa wild-type strain PAO1, an isogenic oxyR mutant with and without a plasmid (pUCP19) control, and a complemented oxyR mutant (poxyR) were used in all experiments described herein. We first employed an acute pneumonia infection model of mice as described previously (11), with the exception that adult rather than infant mice were used. CD-1 mice were infected intranasally with 107 bacteria each. Infected lungs were harvested at 16 h postinfection and homogenized, and serial dilutions were plated for the enumeration of CFU. When infected with wild-type P. aeruginosa, the mice were impaired in their ability to clear the organisms, and the CFU were enumerated at a log of 6.2 (Fig. 1). In contrast, the numbers of CFU of the oxyR mutants without and with the control plasmid pUCP19 were nearly 100-fold lower, at logs of 4.2 and 4.0, respectively (Fig. 1). Importantly, virulence was almost completely restored (5.6 log) in the oxyR mutant harboring poxyR.
We next tested the potential requirement for OxyR for the virulence of P. aeruginosa in a quantitative mouse burn infection model developed by Stevens et al. (10). In this model, a full-thickness burn wound is infected with P. aeruginosa by use of a slight modification of previous methods. First, a skin fold is elevated, and two preheated (92 to 95°C) brass blocks (2 by 2 cm) are applied for 5 s, thereby generating an injury with discrete margins that covers 5% of the total body surface area. With this model, the damaged epidermis and dermis undergo coagulative necrosis, but the underlying rectus abdominis (RA) muscles are not injured. Next, a P. aeruginosa suspension is injected intradermally into the midline crease of the burn eschar. The bacteria then proliferate in the burn wound tissue and invade the normal underlying RA muscles. The number of bacteria found in the RA muscles underlying and adjacent to the initial burn injury after 24 h serves as a sensitive and quantitative measure of local invasiveness. Some strains may also invade the vasculature and lymphatics, resulting in the widespread dissemination of bacteria throughout the infected animal. Hence, any of these organs are appropriate for documenting the systemic spread of P. aeruginosa from the dorsal burn wound. This progression of infection from a local soft-tissue infection caused by the low-dose bacterial inoculum to a systemic dissemination, leading to multisystem organ failure and eventual death, in many ways mimics the sequelae of lethal sepsis in burned or other severely immunocompromised patients.
After infection with 107 bacteria, the titer of wild-type bacteria in the burn eschars increased by 1.6 log after 24 h (Fig. 2). In contrast, there was little or no increase in the CFU counts of oxyR and oxyR (pUCP19) strains (Fig. 2). Importantly, the virulence of the oxyR mutant harboring poxyR was partially restored, with the viable bacterial load increasing by 1.2 log (Fig. 2). We hypothesized that the levels of H2O2 in blood (low micromolar levels) (6) or the levels of H2O2 generated by macrophages of the reticuloendothelial cell system in burned mice will kill the bacteria during their systemic spread. Thus, we examined the abilities of the oxyR mutant to spread systemically to various organs, including the spleen, kidney, lung, and liver, and, in the process, to expose the organisms to blood H2O2. As shown in Fig. 2, the bacterial titers of the oxyR mutant and vector control mutants in various mouse organs were consistently 2 to 3 log lower than the titers of bacteria recovered from identical organs infected with wild-type bacteria. Specifically, the numbers of oxyR mutant bacteria recovered from burn eschar, spleen, kidney, lung, and liver were, respectively, 1.1, 1.9, 2.0, 2.5, and 2.3 log less than the number of bacteria of the wild-type strain, PAO1. Similarly, the numbers of viable oxyR mutant bacteria harboring pUCP19 that were recovered from burn eschar, spleen, kidney, lung, and liver were, respectively, 1.6, 2.4, 2.3, 3.0, and 2.8 log lower than the number of the wild-type bacteria, suggesting that the inclusion of plasmid pUCP19 in trans does not restore the virulence of the oxyR mutant. Significantly, as in the lung infection studies, virulence was partially restored in the oxyR mutant harboring poxyR, and the loads of the viable bacteria in various organs were only 0.5 to 1 log lower than the load of viable wild-type bacteria (Fig. 2).
Neutrophil killing of the oxyR mutant is enhanced relative to that of wild-type bacteria One of the primary cell-mediated defenses against P. aeruginosa is the human neutrophil, especially in the context of cystic fibrosis airway disease. Therefore, we examined whether oxyR mutant bacteria were more sensitive to neutrophil-mediated killing than wild-type bacteria. Each of the four P. aeruginosa strains was incubated for 0 to 3 h in Dulbecco's minimal essential medium in the presence or absence of human neutrophils that had been isolated from the venous blood of normal subjects. At defined times, viable bacteria were measured as numbers of CFU from serial dilutions of each suspension on LB agar containing catalase. As shown in Fig. 3, the oxyR mutant was found to be more sensitive (about 33%) than wild-type bacteria to in vitro killing by human neutrophils. Complementation of poxyR, but not the control plasmid, pUCP19, restored wild-type levels of resistance to neutrophils.
The P. aeruginosa oxyR mutant is impaired in its ability to kill the insect D. melanogaster D. melanogaster has recently been shown to be a valuable alternative to animal models for studies of P. aeruginosa pathogenesis (7). We compared the abilities of P. aeruginosa PAO1 and oxyR mutant bacteria to infect D. melanogaster Oregon-R flies by monitoring fly survival over time. Flies were maintained on a standard yeast-agar-sucrose-corn meal medium at 25°C. Experiments were performed using 4- to 7-day-old adult male flies. The infections were initiated by pricking flies on the dorsal thorax with a 10-μm-diameter needle (Ernest Fullam, Inc., Latham, N.Y.) that had been dipped into a cell suspension containing 107 CFU of PAO1 or oxyR mutant bacteria from an early-stationary-phase bacterial culture/ml (7). Infected flies were kept at 25°C. Flies that died within 12 h after infection (<5%) were not included in mortality determinations. As a control, we established that pricking flies with a sterile needle dipped in a 10 mM concentration of MgSO4 caused no death and that pricked flies quickly recovered from such trauma (Fig. 4).
P. aeruginosa PAO1 is highly lethal to D. melanogaster (7). Survival studies have shown that PAO1-infected flies began to perish at 18 h postinfection and that the infection resulted in the deaths of all flies by 28 h (Fig. 4). In contrast, the mortality of oxyR and oxyR (pUCP19) mutant-infected flies did not occur until 28 h postinfection. Furthermore, the mortality kinetics were consistently delayed for 6 to 10 h in oxyR and oxyR (pUCP19) mutant-infected flies (Fig. 4). Importantly, the virulence of the oxyR mutant complemented with poxyR was equivalent to that of the wild-type strain. Thus, the oxidative killing of the oxyR mutant within Drosophila hemolymph may involve mechanisms similar to those utilized by mammalian hosts.
In summary, the data obtained from the three models of infection indicate that OxyR is important for P. aeruginosa in its establishment of pulmonary infection and burn sepsis, is critical to its systemic spread and survival in the bloodstream, and is required for its virulence in the alternative-model host D. melanogaster. The loss of OxyR-mediated antioxidative mechanisms significantly compromises the organism's ability to survive the onslaught of oxygen radicals produced in infected tissues, blood, and human neutrophils. Based upon the findings of this study, we are currently conducting experiments to identify nontoxic reducing agents that compromise OxyR function, which might eventually lead to a therapeutic agent that would enhance the H2O2-mediated killing of P. aeruginosa during infection.
ACKNOWLEDGMENTS
This work was supported in part by Public Health Service grants AI-40541 (D.J.H.), AI-69845 (D.J.H.), AI-34954 (B.E.B.), U56 AI-057192 (B.E.B.), AI-44642 (B.E.B.), and CA-66081 (B.E.B.) from the National Institutes of Health; a New Technologies Genomics Grant from the Cystic Fibrosis Foundation (D.J.H.); the Research Service of the Department of Veterans Affairs (B.E.B.); and American Lung Association research grant RG-131-N (G.W.L).
All animal studies were performed in accordance with the protocols approved by the Animal Care Committee of the University of Cincinnati College of Medicine.
REFERENCES
1. Borregaard, N., J. M. Heiple, E. R. Simons, and R. A. Clark. 1983. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J. Cell Biol. 97:52-61.
2. Brown, S. M., M. L. Howell, M. L. Vasil, A. J. Anderson, and D. J. Hassett. 1995. Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J. Bacteriol. 177:6536-6544.
3. Hassett, D. J., E. Alsabbagh, K. Parvatiyar, M. L. Howell, R. W. Wilmott, and U. A. Ochsner. 2000. A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J. Bacteriol. 182:4557-4563.
4. Hassett, D. J., and M. S. Cohen. 1989. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3:2574-2582.
5. Howell, M. L., E. Alsabbagh, J.-F. Ma, U. A. Ochsner, M. G. Klotz, T. J. Beveridge, K. M. Blumenthal, E. C. Niederhoffer, R. E. Morris, D. Needham, G. E. Dean, M. A. Wani, and D. J. Hassett. 2000. AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. J. Bacteriol. 182:4545-4556.
6. Lacy, F., D. T. O'Connor, and G. W. Schmid-Schonbein. 1998. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J. Hypertens. 16:291-303.
7. Lau, G. W., B. C. Goumnerov, C. L. Walendziewicz, J. Hewitson, W. Xiao, S. Mahajan-Miklos, R. G. Tompkins, L. A. Perkins, and L. G. Rahme. 2003. The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa. Infect. Immun. 71:4059-4066.
8. Ma, J.-F., U. A. Ochsner, M. G. Klotz, V. K. Nanayakkara, M. L. Howell, Z. Johnson, J. E. Posey, M. L. Vasil, J. J. Monaco, and D. J. Hassett. 1999. Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J. Bacteriol. 181:3730-3742.
9. Ochsner, U. A., M. L. Vasil, E. Alsabbagh, K. Parvatiyar, and D. J. Hassett. 2000. Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182:4533-4544.
10. Stevens, E. J., C. M. Ryan, J. S. Friedberg, R. L. Barnhill, M. L. Yarmush, and R. G. Tompkins. 1994. A quantitative model of invasive Pseudomonas infection in burn injury. J. Burn Care Rehabil. 15:232-235.
11. Tang, H., M. Kays, and A. Prince. 1995. Role of Pseudomonas aeruginosa pili in acute pulmonary infection. Infect. Immun. 63:1278-1285.(Gee W. Lau, Bradley E. Br)
Molecular Genetics, Biochemistry and Microbiology
Division of Pulmonary and Critical Care Medicine, University of Cincinnati College of Medicine
Cincinnati VA Medical Center, Cincinnati, Ohio
TEXT
The role of the H2O2-responsive transactivator OxyR was evaluated in mouse and Drosophila models of Pseudomonas aeruginosa infection and by assays of neutrophil killing. Relative to that of wild-type bacteria, oxyR mutant viability was reduced by 99% or greater in mouse models of acute pneumonia and burn sepsis, and the oxyR mutant was more sensitive to killing by human neutrophils and was delayed in the kinetics of Drosophila melanogaster killing.
During human infection by the opportunistic bacterium Pseudomonas aeruginosa, the organism faces exposure to toxic reactive oxygen intermediates generated by phagocytic cells. During the phagocytic oxidative burst, hydrogen peroxide (H2O2) is generated at high millimolar levels within the phagosomal vacuole (4). Enzymatic defenses against H2O2 in P. aeruginosa are provided by at least three catalases (KatA, KatB, and KatC) (2, 8), several probable peroxidases (refer to www.pseudomonas.com), and four established alkyl hydroperoxide reductases (AhpA, AhpB, AhpCF, and Ohr); members of the last class can also degrade H2O2 and various alkylhydroperoxides (9). The major gene product involved in endogenous H2O2 detoxification in P. aeruginosa is the 170-kDa heterotrimeric catalase KatA (8). Accordingly, katA gene expression is consistently high during vigorous aerobic growth. In fact, KatA activity is maintained at such high levels that even significant H2O2 stress triggers only a twofold increase in expression, suggesting that a high rate of KatA activity is critical for the detoxification of endogenous H2O2 produced during normal aerobic metabolism. In contrast to that of the katA gene, expression of several other oxidative stress defense genes, including katB-ankB (5), ahpB, and ahpCF, is dramatically increased upon exposure to H2O2, organic peroxides, or the redox cycling agent paraquat, suggesting that a highly specific and tightly regulated stress response exists in this organism (5, 9). We have found that such a response is governed by the 34-kDa H2O2-responsive transactivator known as OxyR in P. aeruginosa (9). P. aeruginosa lacking OxyR is exquisitely susceptible to H2O2, even though it possesses wild-type catalase activity (9). In fact, isolated colonies of oxyR mutant bacteria do not even appear on aerobic Luria-Bertani (LB) agar, because autoxidizable components in the medium itself generate 1.2 μM H2O2/min (3). This concentration of H2O2 has been detected in peripheral blood from human donors and is sufficient to kill these organisms (3).
In this study, we tested the hypothesis that OxyR is required for the full virulence of P. aeruginosa by use of (i) a mouse intranasal model of acute pneumonia, (ii) a mouse burn sepsis model, (iii) an in vitro model of neutrophil killing, and (iv) a Drosophila melanogaster alternative model for animal infection.
The P. aeruginosa oxyR mutant is impaired in causing pneumonia and infecting burn wounds in mice. P. aeruginosa wild-type strain PAO1, an isogenic oxyR mutant with and without a plasmid (pUCP19) control, and a complemented oxyR mutant (poxyR) were used in all experiments described herein. We first employed an acute pneumonia infection model of mice as described previously (11), with the exception that adult rather than infant mice were used. CD-1 mice were infected intranasally with 107 bacteria each. Infected lungs were harvested at 16 h postinfection and homogenized, and serial dilutions were plated for the enumeration of CFU. When infected with wild-type P. aeruginosa, the mice were impaired in their ability to clear the organisms, and the CFU were enumerated at a log of 6.2 (Fig. 1). In contrast, the numbers of CFU of the oxyR mutants without and with the control plasmid pUCP19 were nearly 100-fold lower, at logs of 4.2 and 4.0, respectively (Fig. 1). Importantly, virulence was almost completely restored (5.6 log) in the oxyR mutant harboring poxyR.
We next tested the potential requirement for OxyR for the virulence of P. aeruginosa in a quantitative mouse burn infection model developed by Stevens et al. (10). In this model, a full-thickness burn wound is infected with P. aeruginosa by use of a slight modification of previous methods. First, a skin fold is elevated, and two preheated (92 to 95°C) brass blocks (2 by 2 cm) are applied for 5 s, thereby generating an injury with discrete margins that covers 5% of the total body surface area. With this model, the damaged epidermis and dermis undergo coagulative necrosis, but the underlying rectus abdominis (RA) muscles are not injured. Next, a P. aeruginosa suspension is injected intradermally into the midline crease of the burn eschar. The bacteria then proliferate in the burn wound tissue and invade the normal underlying RA muscles. The number of bacteria found in the RA muscles underlying and adjacent to the initial burn injury after 24 h serves as a sensitive and quantitative measure of local invasiveness. Some strains may also invade the vasculature and lymphatics, resulting in the widespread dissemination of bacteria throughout the infected animal. Hence, any of these organs are appropriate for documenting the systemic spread of P. aeruginosa from the dorsal burn wound. This progression of infection from a local soft-tissue infection caused by the low-dose bacterial inoculum to a systemic dissemination, leading to multisystem organ failure and eventual death, in many ways mimics the sequelae of lethal sepsis in burned or other severely immunocompromised patients.
After infection with 107 bacteria, the titer of wild-type bacteria in the burn eschars increased by 1.6 log after 24 h (Fig. 2). In contrast, there was little or no increase in the CFU counts of oxyR and oxyR (pUCP19) strains (Fig. 2). Importantly, the virulence of the oxyR mutant harboring poxyR was partially restored, with the viable bacterial load increasing by 1.2 log (Fig. 2). We hypothesized that the levels of H2O2 in blood (low micromolar levels) (6) or the levels of H2O2 generated by macrophages of the reticuloendothelial cell system in burned mice will kill the bacteria during their systemic spread. Thus, we examined the abilities of the oxyR mutant to spread systemically to various organs, including the spleen, kidney, lung, and liver, and, in the process, to expose the organisms to blood H2O2. As shown in Fig. 2, the bacterial titers of the oxyR mutant and vector control mutants in various mouse organs were consistently 2 to 3 log lower than the titers of bacteria recovered from identical organs infected with wild-type bacteria. Specifically, the numbers of oxyR mutant bacteria recovered from burn eschar, spleen, kidney, lung, and liver were, respectively, 1.1, 1.9, 2.0, 2.5, and 2.3 log less than the number of bacteria of the wild-type strain, PAO1. Similarly, the numbers of viable oxyR mutant bacteria harboring pUCP19 that were recovered from burn eschar, spleen, kidney, lung, and liver were, respectively, 1.6, 2.4, 2.3, 3.0, and 2.8 log lower than the number of the wild-type bacteria, suggesting that the inclusion of plasmid pUCP19 in trans does not restore the virulence of the oxyR mutant. Significantly, as in the lung infection studies, virulence was partially restored in the oxyR mutant harboring poxyR, and the loads of the viable bacteria in various organs were only 0.5 to 1 log lower than the load of viable wild-type bacteria (Fig. 2).
Neutrophil killing of the oxyR mutant is enhanced relative to that of wild-type bacteria One of the primary cell-mediated defenses against P. aeruginosa is the human neutrophil, especially in the context of cystic fibrosis airway disease. Therefore, we examined whether oxyR mutant bacteria were more sensitive to neutrophil-mediated killing than wild-type bacteria. Each of the four P. aeruginosa strains was incubated for 0 to 3 h in Dulbecco's minimal essential medium in the presence or absence of human neutrophils that had been isolated from the venous blood of normal subjects. At defined times, viable bacteria were measured as numbers of CFU from serial dilutions of each suspension on LB agar containing catalase. As shown in Fig. 3, the oxyR mutant was found to be more sensitive (about 33%) than wild-type bacteria to in vitro killing by human neutrophils. Complementation of poxyR, but not the control plasmid, pUCP19, restored wild-type levels of resistance to neutrophils.
The P. aeruginosa oxyR mutant is impaired in its ability to kill the insect D. melanogaster D. melanogaster has recently been shown to be a valuable alternative to animal models for studies of P. aeruginosa pathogenesis (7). We compared the abilities of P. aeruginosa PAO1 and oxyR mutant bacteria to infect D. melanogaster Oregon-R flies by monitoring fly survival over time. Flies were maintained on a standard yeast-agar-sucrose-corn meal medium at 25°C. Experiments were performed using 4- to 7-day-old adult male flies. The infections were initiated by pricking flies on the dorsal thorax with a 10-μm-diameter needle (Ernest Fullam, Inc., Latham, N.Y.) that had been dipped into a cell suspension containing 107 CFU of PAO1 or oxyR mutant bacteria from an early-stationary-phase bacterial culture/ml (7). Infected flies were kept at 25°C. Flies that died within 12 h after infection (<5%) were not included in mortality determinations. As a control, we established that pricking flies with a sterile needle dipped in a 10 mM concentration of MgSO4 caused no death and that pricked flies quickly recovered from such trauma (Fig. 4).
P. aeruginosa PAO1 is highly lethal to D. melanogaster (7). Survival studies have shown that PAO1-infected flies began to perish at 18 h postinfection and that the infection resulted in the deaths of all flies by 28 h (Fig. 4). In contrast, the mortality of oxyR and oxyR (pUCP19) mutant-infected flies did not occur until 28 h postinfection. Furthermore, the mortality kinetics were consistently delayed for 6 to 10 h in oxyR and oxyR (pUCP19) mutant-infected flies (Fig. 4). Importantly, the virulence of the oxyR mutant complemented with poxyR was equivalent to that of the wild-type strain. Thus, the oxidative killing of the oxyR mutant within Drosophila hemolymph may involve mechanisms similar to those utilized by mammalian hosts.
In summary, the data obtained from the three models of infection indicate that OxyR is important for P. aeruginosa in its establishment of pulmonary infection and burn sepsis, is critical to its systemic spread and survival in the bloodstream, and is required for its virulence in the alternative-model host D. melanogaster. The loss of OxyR-mediated antioxidative mechanisms significantly compromises the organism's ability to survive the onslaught of oxygen radicals produced in infected tissues, blood, and human neutrophils. Based upon the findings of this study, we are currently conducting experiments to identify nontoxic reducing agents that compromise OxyR function, which might eventually lead to a therapeutic agent that would enhance the H2O2-mediated killing of P. aeruginosa during infection.
ACKNOWLEDGMENTS
This work was supported in part by Public Health Service grants AI-40541 (D.J.H.), AI-69845 (D.J.H.), AI-34954 (B.E.B.), U56 AI-057192 (B.E.B.), AI-44642 (B.E.B.), and CA-66081 (B.E.B.) from the National Institutes of Health; a New Technologies Genomics Grant from the Cystic Fibrosis Foundation (D.J.H.); the Research Service of the Department of Veterans Affairs (B.E.B.); and American Lung Association research grant RG-131-N (G.W.L).
All animal studies were performed in accordance with the protocols approved by the Animal Care Committee of the University of Cincinnati College of Medicine.
REFERENCES
1. Borregaard, N., J. M. Heiple, E. R. Simons, and R. A. Clark. 1983. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J. Cell Biol. 97:52-61.
2. Brown, S. M., M. L. Howell, M. L. Vasil, A. J. Anderson, and D. J. Hassett. 1995. Cloning and characterization of the katB gene of Pseudomonas aeruginosa encoding a hydrogen peroxide-inducible catalase: purification of KatB, cellular localization, and demonstration that it is essential for optimal resistance to hydrogen peroxide. J. Bacteriol. 177:6536-6544.
3. Hassett, D. J., E. Alsabbagh, K. Parvatiyar, M. L. Howell, R. W. Wilmott, and U. A. Ochsner. 2000. A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J. Bacteriol. 182:4557-4563.
4. Hassett, D. J., and M. S. Cohen. 1989. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. FASEB J. 3:2574-2582.
5. Howell, M. L., E. Alsabbagh, J.-F. Ma, U. A. Ochsner, M. G. Klotz, T. J. Beveridge, K. M. Blumenthal, E. C. Niederhoffer, R. E. Morris, D. Needham, G. E. Dean, M. A. Wani, and D. J. Hassett. 2000. AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. J. Bacteriol. 182:4545-4556.
6. Lacy, F., D. T. O'Connor, and G. W. Schmid-Schonbein. 1998. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J. Hypertens. 16:291-303.
7. Lau, G. W., B. C. Goumnerov, C. L. Walendziewicz, J. Hewitson, W. Xiao, S. Mahajan-Miklos, R. G. Tompkins, L. A. Perkins, and L. G. Rahme. 2003. The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa. Infect. Immun. 71:4059-4066.
8. Ma, J.-F., U. A. Ochsner, M. G. Klotz, V. K. Nanayakkara, M. L. Howell, Z. Johnson, J. E. Posey, M. L. Vasil, J. J. Monaco, and D. J. Hassett. 1999. Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J. Bacteriol. 181:3730-3742.
9. Ochsner, U. A., M. L. Vasil, E. Alsabbagh, K. Parvatiyar, and D. J. Hassett. 2000. Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J. Bacteriol. 182:4533-4544.
10. Stevens, E. J., C. M. Ryan, J. S. Friedberg, R. L. Barnhill, M. L. Yarmush, and R. G. Tompkins. 1994. A quantitative model of invasive Pseudomonas infection in burn injury. J. Burn Care Rehabil. 15:232-235.
11. Tang, H., M. Kays, and A. Prince. 1995. Role of Pseudomonas aeruginosa pili in acute pulmonary infection. Infect. Immun. 63:1278-1285.(Gee W. Lau, Bradley E. Br)