Cytochrome c 4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c 1 and c 5 promote intracellular infection

Abstract

A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c1 and cycB mutants lacking cytochrome c5 had normal siderophore expression, cyc4 mutants defective for cytochrome c4 completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c4 in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c5 in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.

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

“…Recent studies have identified both LbtU as a novel, TonB-independent receptor for legiobactin and LbtC as an inner membrane, ABC-type permease that helps mediate legiobactin assimilation (13,21). We also demonstrated that cytochrome c 4 is required for optimal siderophore production, although the means by which this cytochrome promotes legiobactin expression are unclear (22). Despite these various advances in our understanding of the genetics and membrane transport of legiobactin, elucidation of the structure of the L. pneumophila siderophore has remained elusive.…”

The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila

“…DRelative growth on non-iron-supplemented BCYE agar containing 10-14 mM DFX. dData previously reported (Allard et al, 2006;Yip et al, 2011). §+, Ability to utilize legiobactin-containing supernatants for growth stimulation on non-iron-supplemented BCYE agar containing 400 mM DIP at 25 u C. ||Unexplained variable phenotype.…”

“…These mutants (listed in Table 1 by numbers ranging from NU205 to NU244) had been initially identified based upon their hypersensitivity to the iron chelator EDDA in their growth media and/or their resistance to streptonigrin, an antibiotic whose toxicity requires significant levels of intracellular iron (data summarized in Table 3). Subsequent studies found that none of these mutants is impaired for the production of legiobactin (summarized in Table 3), with the exception of NU208, which had proved to contain an insertion in the cytochrome c maturation locus (Table 1) (Allard et al, 2006;Yip et al, 2011). Continuing the genetic analysis that had begun with NU208, NU216 and NU244 (Viswanathan et al, 2000(Viswanathan et al, , 2002, we performed inverse PCR and DNA sequence analysis and defined the site of the mini-Tn10 insertion in most of the ira mutants, i.e.…”

Legionella Pathogenesis and Virulence Factors

“…In addition, LbtU, LbtC and Cyc4 also play roles in iron acquisition. Cyc4 promote secretion of the legiobactin, LbtU and LbtC with their coded genes, lbtU and lbtC mediate uptake of Fe3+ legiobactin complexes in which LbtU, as a TonBindependent receptor for the siderophore specifically promotes L. pneumophila's utilization of legiobactin and likely acts as the receptor for the Legionella siderophore, LbtC involves in iron regulated [81,[83][84][85].…”