Iron availability, oxygen limitation, Pseudomonas aeruginosa and cystic fibrosis

A.-P. Zeng and E.-J. Kim

GBF – Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany

Correspondence
A.-P. Zeng
(AZE{at}GBF.de)

We appreciate the comments of Reid & Kirov (2004) on our recent study (Kim et al., 2003) and their general comments on the issue of iron availability in the lung of cystic fibrosis (CF) patients. By referring to a recent publication from the same group (Reid et al., 2002), these authors argued that the CF lung is not iron deplete but rather the opposite situation exists. We read with interest the publication of Reid et al. (2002) and the other two relevant articles (Stites et al., 1998, 1999) cited by Reid & Kirov (2004) as support for their conjecture. It is noted that Reid et al. (2002) and Stites et al. (1998, 1999) merely measured the total iron concentration (including both free iron and iron bound to iron-binding proteins such as ferritin and transferrin) in sputum samples of CF patients. In our opinion, it is important to distinguish between the total iron concentration in sputum and iron that is really available for Pseudomonas aeruginosa at the site of infection. In addressing this question, several other factors should be considered. First, in an aerobic environment, iron exists mainly as Fe3+ that is almost insoluble and thus less usable for micro-organisms unless iron-chelating agents are present or iron-binding proteins such as siderophores are produced by the cells (Ratledge & Dover, 2000). The local oxygen concentration at the infection site can be therefore decisive for the iron availability. Second, iron-binding proteins in the sputum may exist in an iron-unsaturated form as observed for lactoferrin, a defence protein generated by the host cell to limit bacterial growth by sequestering iron (Thompson et al., 1990; Singh et al., 2002). Thus, the free iron concentration may be very low despite a relatively high overall iron concentration. Third, it is known that P. aeruginosa exists mainly as biofilm in the CF lung (Singh et al., 2000; Costerton et al., 1999). For iron uptake by P. aeruginosa, iron has to be transported into the biofilm. Thus, simply measuring the total iron concentration in the sputum as done in the work of Reid et al. (2002) cannot give a conclusive judgment about the iron availability of P. aeruginosa infecting the CF lung. In fact, Frederick et al. (2001) showed that biofilms of P. aeruginosa exhibit typical behaviour of iron starvation. The results of Frederick et al. (2001) are consistent with previous observations in clinical isolates that P. aeruginosa in the CF lung grows under iron-restricted conditions (Brown et al., 1984; Haas et al., 1991). Our in vitro study (Kim et al., 2003) showed a significant increase of siderophore formation in P. aeruginosa under iron limitation. This correlates with the observed increase of siderophore formation in sputa of CF patients (Haas et al., 1991). In fact, Reid et al. (2002) also confirmed the prevalence of iron deficiency in serum of CF patients. They showed that this iron deficiency is directly related to the increased severity of suppurative lung disease.

Irrespective of the question of whether an iron deficiency prevails in the CF lung, the results of our recent studies (Sabra et al., 2002; Kim et al., 2003) with respect to oxygen transfer in P. aeruginosa culture are relevant to P. aeruginosa infection in the CF lung in view of the recent discovery that the microbial environment of the CF lung is essentially anaerobic or hypoxic (Worlitzsch et al., 2002; Yoon et al., 2002). Worlitzsch et al. (2002) postulated that an increased O2 consumption rate of CF lung epithelial cells may initially cause an O2 gradient in the thickened mucus mass of the CF lung airway which gradually becomes severely hypoxic after infection by P. aeruginosa due to additional O2 consumption of the bacteria. Our results indicate that the strongly reduced O2 transfer rate caused by P. aeruginosa itself may also significantly contribute to the occurrence of oxygen limitation, especially when iron deficiency prevails in the mucus of CF airways. By generating an anaerobic environment, the solubility and thus the availability of iron can be significantly increased. In this way, more iron may be trapped in iron-binding proteins, giving a possible explanation for the increased overall iron concentration in CF sputum as observed by Reid et al. (2002) and Stites et al. (1998, 1999). A lower oxygen tension is also important for reducing the formation of oxidative radicals by the innate immune system of host cells. Thus, the blockage of O2 transfer rate triggered by iron limitation as observed in vitro may represent an efficient tactic of this pathogen to deal with iron deficiency and host defence and deserves further investigations in connection with CF lung infection by P. aeruginosa.

In addition to causing oxygen limitation, iron deficiency can have further implications for the development of P. aeruginosa biofilm and its treatment. For example, Scharfman et al. (1996) reported that the adhesion of P. aeruginosa to respiratory mucins and the expression of mucin-binding proteins are increased by iron limitation during growth. More important, the biofilm of a clinical isolate of P. aeruginosa was shown to be very resistant to the antibiotic tobramycin when grown in vitro under iron limitation (Anwar et al., 1989).

Of course, the physiological responses reported for P. aeruginosa PAO1 grown in vitro should be examined with more clinically relevant strains, ideally under in vivo conditions. As to the question of whether PAO1 should be used at all for addressing questions regarding P. aeruginosa infection, we think that the comments of Reid & Kirov are somewhat biased. Although genotypic and phenotypic variations of P. aeruginosa are often observed in strains isolated from different environments, there exist some common fundamental manifestations of this bacterium in their physiological responses to the change of environmental conditions. Quorum-sensing is a good example for this. In fact, without in-depth in vitro studies with PAO1 and other P. aeruginosa strains under well-defined experimental conditions, many of the important concepts that have greatly improved our understanding of P. aeruginosa infection and pathogenicity such as biofilm formation, quorum-sensing and mechanisms of virulence factor formation would have not evolved to their present maturity. In this connection, it is worth mentioning the use of PAO1 in addition to clinical isolates in the studies of Singh et al. (2000, 2002) and Worlitzsch et al. (2002) that are mentioned above and, in our opinion, represent important recent advances in the field of P. aeruginosa infection of CF patients.

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