Bacterial Siderophores That Evade or Overwhelm Lipocalin 2 Induce Hypoxia Inducible Factor 1α and Proinflammatory Cytokine Secretion in Cultured Respiratory Epithelial Cells

Abstract

e Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent؉Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent؉Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1␣ protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1␣ (HIF-1␣), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1␣ was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.

Siderophores: More than Stealing Iron

“…Both phenotypes were independent of lipocalin-2 expression, as similar levels of pathogen dissemination and host expression of proinflammatory cytokines were observed in both wild-type and lipocalin-2-deficient mice. This is in contrast to prior in vitro observations, where lipocalin-2 enhanced production of cytokines in airway epithelial cells (16), a difference that Holden et al suggested might be the result of redundant signals compensating for lipocalin-2 deficiency in vivo .…”

“…It was previously shown that siderophores secreted by enteric pathogens cause hypoxia-dependent activation of HIF-1α in the Peyer’s patches and in human epithelial and endothelial cells (15). Previously, Holden et al demonstrated that enterobactin stabilizes HIF-1α in respiratory cells in vitro , thereby inducing expression of proinflammatory cytokines and enhancing lipocalin-2-mediated inflammation (16). In a more recently published mBio article by Holden et al (17), the authors hypothesized that siderophores secreted by K. pneumoniae during lung infection can also have proinflammatory effects by interacting with host cells, thereby promoting pathogenicity during pneumonia.…”

Oxygen metabolism and barrier regulation in the intestinal mucosa

“…Pathogenic gram-negative bacteria acquire iron from the host through the secretion of siderophores, which chelate iron for incorporation into normal biological processes. Holden et al demonstrated that bacterial siderophores such as enterobactin chelate iron to the extent that HIF is stabilized and HIF target genes are induced (94). Likewise, it was shown that siderophores from a number of bacterial genera (e.g., Salmonella, Yersenia, Enterobacter) are capable of stabilizing HIF (95 (110).…”

Siderophore-based immunization strategy to inhibit growth of enteric pathogens

“…Broadly, our work indicates that new preventive and therapeutic strategies for microbial infections should target siderophore-mediated iron acquisition, a virulence trait shared by almost all bacterial and fungal pathogens (9). Moreover, because recent studies demonstrate additional, nonclassical roles for siderophores (46,47), including protection of bacteria from oxidative stress (48) and modulation of host gene expression (49), it is possible that antibody-mediated sequestration of siderophores may inhibit bacterial growth by iron-independent mechanisms and may provide additional benefits to the host.…”