The Legionella pneumophila Siderophore Legiobactin Is a Polycarboxylate That Is Identical in Structure to Rhizoferrin

Abstract

bLegionella pneumophila, the agent of Legionnaires' disease, secretes a siderophore (legiobactin) that promotes bacterial infection of the lung. In past work, we determined that cytoplasmic LbtA (from Legiobactin gene A) promotes synthesis of legiobactin, inner membrane LbtB aids in export of the siderophore, and outer membrane LbtU and inner membrane LbtC help mediate ferrilegiobactin uptake and assimilation. However, the past studies examined legiobactin contained within bacterial culture supernatants. By utilizing high-pressure liquid chromatography that incorporates hydrophilic interaction-based chemistry, we have now purified legiobactin from supernatants of virulent strain 130b that is suitable for detailed chemical analysis. High-resolution mass spectrometry (MS) revealed that the molecular mass of (protonated) legiobactin is 437.140 Da. On the basis of the results obtained from both MS analysis and various forms of nuclear magnetic resonance, we found that legiobactin is composed of two citric acid residues linked by a putrescine bridge and thus is identical in structure to rhizoferrin, a polycarboxylate-type siderophore made by many fungi and several unrelated bacteria. Both purified legiobactin and rhizoferrin obtained from the fungus Cunninghamella elegans were able to promote Fe 3؉ uptake by wild-type L. pneumophila as well as enhance growth of iron-starved bacteria. These results did not occur with 130b mutants lacking lbtU or lbtC, indicating that both endogenously made legiobactin and exogenously derived rhizoferrin are assimilated by L. pneumophila in an LbtU-and LbtC-dependent manner.T he Gram-negative bacterium Legionella pneumophila is both an inhabitant of natural and man-made water systems and the primary etiologic agent of Legionnaires' disease, an increasingly common and serious form of pneumonia (1-3). In its aquatic habitats, L. pneumophila survives in biofilms and as an intracellular parasite of amoebae, and in the lung, it grows primarily in macrophages (4-7). Iron has long been recognized as a major aspect of L. pneumophila replication, intracellular infection, and virulence (8, 9). For ferrous iron assimilation, L. pneumophila utilizes a secreted pyomelanin that has ferric reductase activity and the inner membrane Fe 2ϩ transporter FeoB (10, 11). For ferric iron uptake, the bacterium uses the siderophore legiobactin (12, 13). Both of these iron acquisition pathways are required for infection of the lungs by L. pneumophila (10,14).The investigation of legiobactin has followed a rather circuitous path. Indeed, it was originally thought that L. pneumophila does not produce siderophores; this conclusion was based on negative data obtained from both the Arnow and Csáky assays, which detect catecholate and hydroxymate structures, respectively, and the Chrome Azurol S (CAS) assay, which detects iron chelators independently of their structure (15-17). However, in 2000, we demonstrated that L. pneumophila strains can secrete a siderophore activity that is detected by the CAS assay when the ba...

Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila

“…Consistent with the behavior of the ⌬lbtU and ⌬lbtP single mutants, the ⌬lbtU ⌬lbtP double mutant also was unable to utilize iron-loaded siderophore to support growth on iron-depleted media. Recently, it was determined that legiobactin is equivalent in structure to rhizoferrin, a siderophore first identified in fungi (27). Using purified rhizoferrin as the sole iron source, similar results were obtained (see Fig.…”

“…In E. coli, FlxA is a member of the flagellar regulon but is dispensable for motility (44). The C-terminal do- (45) that imports the iron-bound siderophore legiobactin (27,29). The genes responsible for siderophore production and transport in the L. pneumophila 130b strain are conserved in the L. pneumophila Philadelphia-1 strain (lpg1323-lpg1326) (23,27,29,30) (Fig.…”

“…The ability of L. pneumophila to acquire iron from the host cell is a critical determinant in the outcome of infection (19). As a consequence, L. pneumophila employs several mechanisms for iron acquisition and assimilation during intracellular growth in cultured macrophages and a lung infection model (20)(21)(22)(23)(24)(25)(26)(27). One mechanism employs legiobactin, an iron-chelating siderophore that has been extensively characterized in the L. pneumophila strain 130b (28).…”

“…The plates were incubated at 37°C for 8 days, and growth of bacteria was compared. For rhizoferrin utilization experiments, L. pneumophila strains were plated on non-iron-supplemented CYE medium containing 8 M DFX, and 50 l of 40 g/ml of purified rhizoferrin (EMC Microcollections, Tubingen, Germany) was added to wells cut in the center of the agar plate as previously described (27).…”

Legionellashows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase

“…From Legionella , only four BGCs have been explored in detail with three secondary metabolites structurally elucidated to date (Fig. 1) (Ahrendt et al, 2013; Shevchuk et al, 2014; Burnside et al, 2015; Johnston et al, 2016a). Legioliulin (1), a product of a trans -AT PKS cluster first identified in L. dumoffii, was reported originally in 2004 (Amemura-Maekawa et al, 2004) and biological activity assays failed to determine a role for the compound beyond fluorescence (Ahrendt et al, 2013).…”