Abstract
Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.
Highlights
The battle for iron between host and pathogen has shaped both mammalian nutritional immunity and microbial pathogenesis (Cassat and Skaar, 2013)
The genetic structure of the hmuRSTUV hemin uptake locus has been characterized in Yersinia pestis (Thompson et al, 1999)
We extend this study by using RNA-seq analysis in the related pathogen Y. pseudotuberculosis to measure expression of hmuRSTUV under conditions when cells had been depleted of iron or when inorganic iron or hemin were added back as iron sources after a period of iron starvation (Figures 2B–D, Supplementary Dataset)
Summary
The battle for iron between host and pathogen has shaped both mammalian nutritional immunity and microbial pathogenesis (Cassat and Skaar, 2013). Inflammatory cytokines such as IL-6 induce hypoferremia, further limiting the amount of iron available to invading pathogens Microbial pathogens such as the plague agent Yersinia pestis and the enteropathogens Y. pseudotuberculosis and Y. enterocolitica encode numerous iron acquisition systems (Forman et al, 2010). The cytoplasmic protein HmuS has been proposed to play a role in the detoxification and degradation of hemin, as deletion of the HmuS homolog in Yersinia enterocolitica, HemS, was reported to be lethal (Stojiljkovic and Hantke, 1994) Consistent with this function, studies in Yersinia pseudotuberculosis have shown that HmuS releases iron via degradation of heme to biliverdin in the presence of molecular oxygen, an electron donor such as NADPH, and ferrodoxin-NADP+ reductase (Onzuka et al, 2017). This work increases our understanding of iron control of heme uptake and the role of IscR in Yersinia pseudotuberculosis survival and growth in mammalian tissues
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