JOURNAL OF CLINICAL MICROBIOLOGY, June 2004, p. 2806–2809 0095-1137/04/$08.00⫹0 DOI: 10.1128/JCM.42.6.2806–2809.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 6
Survey of Ferroxidase Expression and Siderophore Production in Clinical Isolates of Pseudomonas aeruginosa Wilhelmina M. Huston,1 Adam J. Potter,1 Michael P. Jennings,1* Jordi Rello,2 Alan R. Hauser,3,4 and Alastair G. McEwan1 Centre for Metals in Biology, Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, 4072 Queensland, Australia1; Department of Microbiology and Immunology3 and Department of Medicine,4 Northwestern University, Chicago, Illinois 60611; and Critical Care Department, Hospital Universitari Joan XXIII, Universitat Rovira i Virgili, Barcelona, Spain2 Received 14 August 2003/Returned for modification 22 October 2003/Accepted 2 December 2003
Ferroxidase (encoded by the mco gene), a component of a ferrous iron uptake pathway in Pseudomonas aeruginosa, was detected in all of the 35 respiratory clinical isolates surveyed; in contrast, considerable variation in siderophore expression was observed. The ubiquitous expression of this periplasmic ferroxidase suggests that it plays a key role in iron uptake in this opportunistic pathogen. that P. aeruginosa possesses additional iron uptake mechanisms that have a role in pathogenesis. Recently, we identified an Fe(II) iron acquisition pathway of P. aeruginosa that is dependent on a periplasmic multicopper oxidase (MCO) and independent of siderophores. This enzyme acts as a ferroxidase, converting Fe(II) to Fe(III) (7). In the host, this may occur after reductive release of iron from transferrin by leukopyocyanin (3). In this paper, we report the examination of 35 isolates of P. aeruginosa with defined clinical outcomes for the presence of the MCO protein and siderophores. Respiratory clinical isolates of P. aeruginosa, Pse 1 to Pse 41, previously assembled by Hauser and coworkers from ventilator-associated pneumonia patients with a variety of clinical outcomes from death-related to complete recovery, were used to conduct the investigation (6). Control strains used for the experiments included P. aeruginosa PAO1 and PAK, siderophore-deficient mutants generated by Ankenbauer and coworkers (IA613, IA602, IA629, and IA635) (1), and an mco Tcr mutant strain (7). The clinical isolates and control strains were examined for the production of pyoverdin and pyocyanin by using King B and King A media, respectively. Quantitation of siderophores was also carried out with the chrome azurol S (CAS) reagent in a spectrophotometric assay (14). The total data collected from the isolates are shown in Table 1. King A and King B solid media were streaked from Luria-Bertani plate cultures of the isolates and incubated overnight at 37°C. The CAS assay was conducted from cell supernatants of the cultures grown for 24 h in CPS medium. CPS medium was prepared with 0.8% Casamino Acids, 5 mM K2SO4, 5 mM K2HPO4, and 5 mM KH2PO4. The solution was autoclaved prior to the addition of MgSO4 to a final concentration of 1 mM. Supernatants were obtained by centrifugation at 4,000 ⫻ g for 10 min, and each isolate was examined in triplicate. Supernatants were incubated with the Fe(III)-loaded CAS reagent for 30 min prior to spectrophotometric determination of the CAS-Fe(III) complex remaining. Medium-only controls were included in the experiment. Pyoverdin production is shown in Table 1. In a qualitative
Pseudomonas aeruginosa is the causative agent for a variety of severe human infections and disease, including colonization of the lungs of cystic fibrosis patients and infection of burns and immunocompromised patients (8). This opportunistic pathogen is found in a diverse range of ecological niches, including soil, plants, and animal hosts. P. aeruginosa has evolved a variety of iron acquisition mechanisms. Although it is required at a physiological concentration of around 10⫺7 to 10⫺5 M in most bacteria, iron is not freely available in the human host. In animals, iron is stored intracellularly as Fe(III) in ferritin, while this ion is transported throughout the body by using the Fe(III) binding proteins, such as transferrin. Iron sequestration via transferrin and lactoferrin is of critical importance in the prevention of iron acquisition by bacteria during infection (21), and recently it was shown that lactoferrin prevents establishment of P. aeruginosa biofilms by preventing bacterial iron acquisition (15). Faced with the lack of freely available iron, pathogenic bacteria have evolved a number of systems to extract iron from the host (2, 21). Most research has focused on the production of siderophores, low-molecular-weight molecules with a high affinity for Fe(III) that are capable of acquiring iron from the host stores. In P. aeruginosa, two siderophores, pyochelin and pyoverdin, have been characterized (19). Pyochelin is considered to play only a minor role during pathogenicity due to its relatively low affinity for Fe(III) and the lack of virulence deficiency of pyochelin mutants in animal model systems (4, 9, 17). Although experiments using the burnt mouse model system have indicated that pyoverdin is essential for virulence of P. aeruginosa (9), isolates lacking pyoverdin have been found in infected cystic fibrosis patients (5). It has also been reported that siderophore-deficient mutants are capable of establishing infection in immunocompromised mice when inoculated both intramuscularly and intranasally (17). These reports suggest * Corresponding author. Mailing address: Centre for Metals in Biology, Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, University of Queensland, St Lucia, 4072, Queensland, Australia. Phone: 61 7 3365 4879. Fax: 61 7 3365 4620. E-mail:
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FIG. 1. Quantitative determination of siderophore production by the clinical isolates. The removal of iron from the CAS reagent by the supernatants of each isolate is expressed by 1/[CAS-FE] on the y axis. 1/[CAS-FE] is the inverse of the remaining reagent with Fe(III) bound, which was determined by the spectrophotometric method, and the molar concentration of CAS-FE was calculated by using the molar extinction coefficient as previously described by Schwyn and Neilands (14). The horizontal line represents the baseline as determined by medium-only controls. The error bars represent 1 standard deviation.
test using King B medium to screen for pyoverdin production under iron-limited conditions, production of pyoverdin appeared to be absent in four of the clinical isolates, one of which was associated with death of the human host. Quantitation of siderophore production using the CAS reagent showed that its level was highly variable in the death-related clinical isolates (Pse 1 to Pse 10) (Fig. 1). In fact, the death-associated isolate Pse 17 had one of the lowest levels of siderophore activity detected in this collection of isolates. Similarly, some of the isolates (Pse 35 and Pse 39), which had low 50% lethal doses (LD50s) as reported by Schulert and coworkers (13) indicating strong virulence in the animal model, had low levels of siderophore activity (Table 1). Taken together, the results show that siderophore production in strains of pathogenic P. aeruginosa is highly variable. This is consistent with previous observations of De Vos et al. (5), who examined isolates of P. aeruginosa but did not report clinical outcomes associated with strains. P. aeruginosa has also been shown to acquire iron from human transferrin in vitro by two distinct mechanisms (3). In addition to the siderophore-dependent removal of Fe(III) from transferrin, it was observed that iron could be mobilized by the redox active phenazine dye, pyocyanin. Reduction of Fe(III) to Fe(II) by leukopyocyanin generates a soluble form of iron that is not bound tightly by transferrin. It was shown by Cox (3) that P. aeruginosa was capable of taking up Fe(II) mobilized by pyocyanin. Recently, we showed that the MCO of P. aeruginosa was important for Fe(II) acquisition under aerobic conditions and hypothesized that it acted as a ferroxidase in a pathway whose existence was first established by Cox (3) The clinical isolates were screened for the presence of the MCO via
Western blotting, using the polyclonal sera previously described in reference 7. The MCO protein was detected in all of the clinical isolates examined during this study (Fig. 2 and Table 1). Given the presence of this protein in all of the clinical isolates examined, we suggest that this iron uptake mechanism may also play an important function during pathogenicity of P. aeruginosa. The acquisition of iron in the host environment by P. aeruginosa has been largely attributed to the siderophore pyoverdin (19, 20). However, the organism is known to possess a number of other important mechanisms, including heme uptake (11)
FIG. 2. Western blot of selected isolates. The figure shows Western blots of total soluble extracts, using polycolonal sera to the MCO (7) (total protein concentration of 1 mg/ml). Lanes in blot 1: 1, Benchmark (Invitrogen); 2, Pse 1; 3, Pse 5; 4, Pse 12; 5, Pse 17; 6, Pse 25; 7, Pse 39; 8, mco Tcr mutant; 9, PAK. Lanes in blot 2: 10, Pse 41; 11, IA613; 12, PAO1; 13, mco Tcr mutant; 14, PAK. The arrowhead indicates the position of the 65.8-kDa molecular mass marker. The marker apparent molecular masses are 179.3, 120, 83.8, 65.8, 50.1, 38.6, 27.5, 20.5, 16.1, and 6.7 kDa.
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TABLE 1. Complete data collected from clinical isolates and reference strains Isolate
Pse 1 Pse 2 Pse 3 Pse 4 Pse 5 Pse 6 Pse 7 Pse 8 Pse 9 Pse 10 Pse 11 Pse 12 Pse 13 Pse 14 Pse 15 Pse 16 Pse 17 Pse 18 Pse 19 Pse 20 Pse 21 Pse 22 Pse 23 Pse 24 Pse 25 Pse 26 Pse 27 Pse 28 Pse 29 Pse 30 Pse 33 Pse 35 Pse 37 Pse 39 Pse 41 PAK PA01 IA613 IA602 IA629 IA635
Clinical outcomea
LD50 105 CFUb
Death, related Death, related Death, related Death, related Death, related Death, related Death, related Death, related Death, related Death, related Death, crude Death, crude Death, crude Death, crude Death, crude Death, crude Death, crude Death, crude Death, crude Recovery Recovery Recovery Recovery Recovery Recovery Recovery Recovery Recovery Recovery Relapse Relapse Relapse Relapse Relapse Relapse
350 509 424 59 354 360 880 742 13 79 78 731 392 89 199 424 78 201 689 201 344 244 689 244 238 689 466 265 199 180 848 79 371 40 159
Presence of: Pyocyanin (King A medium)c
Pyoverdin (King B medium)d
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫺/⫺ ⫹/⫹ ⫹/⫹ ⫹/⫹ ⫺/⫺ ⫺/⫺ ⫺/⫺ ⫺/⫺
a Clinical isolates are described in reference 6. “Death, related” refers to cases where the pulmonary infection was thought to be a contributing factor to the death. “Death, crude” refers to cases where the death occurred after the resolution of the pneumonia. Note that Western blotting showed the presence of MCO protein for each isolate. b LD50 in mouse acute pneumonia model as described in reference 13. c ⫹, visible pyocyanin present; ⫺, no visible pyocyanin. d Detection of pyoverdin is described by the presence or absence of visible color and presence (⫹) or absence (⫺) of fluorescence under UV light.
and degradation and scavenging of iron from host sources such as transferrin (3), and a putative ferrous iron transporter encoded by feoB can be found in the genome sequence (16). There have been some suggestions that the acquisition of heme, for which two separate mechanisms can be found in P. aeruginosa, may compensate in the siderophore-deficient strains (11, 17). The FeoB ferrous iron uptake protein of Legionella pneumophila has been recently reported be important for intracellular infections (12). Similarly, the FeoB protein of Salmonella enterica serovar Typhimurium was also shown to be important during colonization of the intestine of mice (18). The uptake of Fe(II) under aerobic conditions during patho-
genicity via a combination of redox molecules and a ferroxidase-dependent iron uptake system (10) appears to be an emerging and important area of bacterial iron acquisition. The identification of this novel MCO-dependent ferrous iron acquisition system in P. aeruginosa and its presence in all examined clinical isolates, despite highly varied siderophore levels, strongly indicate a likely function for this system during pathogenicity. Furthermore, the presence of homologous putative MCOs in many of the other gram-negative pathogen genomes (7) suggests that ferroxidase-dependent iron uptake systems may be of widespread importance. W.M.H. is supported by an Australian Postgraduate Award and was also the recipient of the UQ Graduate School Research Travel Award. J.R.’s research on Pseudomonas is currently sponsored by REDRESPIRA (RTICC/13), CIRIT (SGR 2000/128), and Distincio a la Recerca Universitaria. Research on iron acquisition in the laboratories of M.P.J. and A.G.M. is funded by NHMRC grant no. 252885. We would like to acknowledge Dolors Mariscal for her contributions in collecting the strains. REFERENCES 1. Ankenbauer, R. G., A. L. Staley, K. L. Rinehart, and C. D. Cox. 1991. Mutasynthesis of siderophore analogues by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 88:1878–1882. 2. Braun, V. 2001. Iron uptake mechanisms and their regulation in pathogenic bacteria. Int. J. Med. Microbiol. 291:67–79. 3. Cox, C. D. 1986. Role of pyocyanin in the acquisition of iron from transferrin. Infect. Immun. 52:263–270. 4. Cox, C. D., K. L. Rinehart, M. L. Moore, and J. C. Cook. 1981. Pyochelin: novel structure of an iron-chelating growth promoter from Pseudomonas aeruginosa strains. Proc. Natl. Acad. Sci. USA 78:302–308. 5. De Vos, D., M. De Chial, C. Cochez, S. Jansen, B. Tummler, J. M. Meyer, and P. Cornelis. 2001. Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations. Arch. Microbiol. 175:384–388. 6. Hauser, A. R., E. Cobb, M. Bodi, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30:521–528. 7. Huston, W. M., M. P. Jennings, and A. G. McEwan. 2002. The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition. Mol. Microbiol. 45:1741–1750. 8. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microb. Infect. 2:1051–1060. 9. Meyer, J.-M., A. Neely, A. Stintzi, C. Georges, and I. A. Holder. 1996. Pyoverdin is essential for virulence of Pseudomonas aeruginosa. Infect. Immun. 64:518–523. 10. Newman, D. K., and R. Kolter. 2000. A role for excreted quinones in extracellular electron transfer. Nature 405:94–97. 11. Ochsner, U. A., Z. Johnson, and M. L. Vasil. 2000. Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa. Microbiology 146:185–198. 12. Robey, M., and N. P. Cianciotto. 2002. Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection. Infect. Immun. 70: 5659–5669. 13. Schulert, G. S., H. Feltman, S. D. P. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser. 2003. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates from patients with hospital acquired pneumonia. J. Infect. Dis. 188:1695–1706. 14. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47–56. 15. Singh, P. K., M. R. Parsek, P. E. Greenberg, and M. J. Welsh. 2002. A component of innate immunity prevents bacterial biofilm development. Nature 417:552–555. 16. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, and I. T. Paulsen. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964. 17. Takase, H., H. Nitanai, K. Hoshino, and T. Otani. 2000. Impact of sidero-
VOL. 42, 2004 phore production on Pseudomonas aeruginosa infections in immunosuppressed mice. Infect. Immun. 68:1834–1839. 18. Tsolis, R. M., A. J. Baumler, F. Heffron, and I. Stojiljkovic. 1996. Contribution of TonB- and Feo-mediated iron uptake to growth of Salmonella typhimurium in the mouse. Infect. Immun. 64:4549–4556. 19. Vasil, M. L., and U. A. Ochsner. 1999. The response of Pseudomonas aerugi-
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