Discovery of a Nonclassical Siderophore, Legiobactin, Produced by Strains ofLegionella pneumophila

Abstract

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 M iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.The bacterium Legionella pneumophila is a ubiquitous inhabitant of natural and man-made aquatic environments, surviving both free, in biofilms, and as an intracellular parasite of protozoa (1,8,26,49,50). Yet, this gram-negative microbe is best known for being the principal etiologic agent of Legionnaires' disease, a potentially fatal form of human pneumonia (59,95). Within the lung, L. pneumophila flourishes as an intracellular parasite of the alveolar macrophages and perhaps the epithelium (1,8,13,41,63,84). A variety of studies indicate that iron is a key requirement for L. pneumophila extracellular replication, intracellular infection, and virulence (7,9,10,25,31,39,40,44,45,60,65,71,72,76,77,78,86,89,91). Despite this, the molecular basis of Legionella iron acquisition, particularly its earliest stages, is relatively unclear.Among all of the considerations regarding Legionella iron acquisition, it is the issue of siderophore production by L. pneumophila that has been the most controversial. In the early 1980s, shortly after the discovery of the Legionella genus, it was reported that L. pneumophila does not produce siderophores (79). This conclusion was based upon the negative results that were obtained from a standard bioassay, as well as the Arnow and Csáky assays, the customary methods for detecting ...

Analysis of a DtxR-Regulated Iron Transport and Siderophore Biosynthesis Gene Cluster in Corynebacterium diphtheriae

“…Another unusual feature of this siderophore system is that while CiuE has homology to enzymes involved in the synthesis of aerobactin, a hydroxamate siderophore, the C. diphtheriae siderophore gives a negative result in tests for the hydroxamate-type siderophores (27). Similar observations have been made in studies with siderophores produced by Bacillus anthracis (6) and Legionella pneumophila (15,19). Synthesis of the siderophore anthrachelin in B. anthracis appears to require two genes that share homology to iucC and iucA, yet anthrachelin gives a negative result in the Csaky test for hydroxamates.…”

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

“…In order to assess legiobactin production, secretion, and utilization, L. pneumophila strains were grown in deferrated CDM at 37°C, and then at 24 and 48 h postinoculation, cell-free culture supernatants were obtained as described previously (2,3). Siderophore activity within the supernatants was quantified using the CAS assay as previously done, with DFX serving as the standard (2,3,84,123). Supernatants were tested for siderophore biological activity by examining their ability to promote the growth of the NU269 feoB mutant on non-iron-supplemented BCYE agar (2,3).…”

“…To monitor the basic extracellular growth capacity of L. pneumophila, bacteria grown on BCYE agar were inoculated into buffered yeast extract (BYE) or CDM broth, and then the optical densities at 660 nm (OD 660 ) of the cultures were determined (62,84,108,132). BYE and CDM broths also typically contain the iron supplement.…”

“…The principal means of L. pneumophila Fe 3ϩ assimilation is legiobactin. When L. pneumophila is grown in a low-iron, chemically defined medium (CDM), it secretes this low-molecular-weight siderophore, which is most readily detected by the chrome azurol S (CAS) assay (3,84). Legiobactin is also able to stimulate the growth of iron-starved legionellae, including the wild type and a feoB mutant (2,3).…”

Production of yellow-green fluorescent pigment by Pseudomonas fluorescens

“…These facts explain why iron is not readily available to microorganisms. Microorganisms other than Pseudomonas like Legionella pneumophila (Liles et al, 2000), Halomonas, Marinobacter (Martinez et al, 2000), and Rhizobium (Guerinot et al, 1990;Barsomian et al, 1992;Persmark et al, 1993;Roy et al, 1994), can produce iron-scavenging molecules. As microorganisms are in competition for available nutrients, including iron, it is not uncommon to link iron-binding molecules to microbial virulence (Sokol, 1986;Takase et al, 2000;Lamont et al, 2002).…”