Journal
MOLECULAR MICROBIOLOGY
Volume 80, Issue 6, Pages 1581-1597Publisher
WILEY
DOI: 10.1111/j.1365-2958.2011.07667.x
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Funding
- NIH [GM53836]
- NSF [MCB0417694]
- CNRS
- INSERM
- Universite Paris Descartes
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1316251] Funding Source: National Science Foundation
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We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putativeABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [Fe-59]-Hn transport, and purified Lmo2185 bound [Fe-59]-Hn (KD = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a haemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [Fe-59]-Hn transport at external concentrations less than 50 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (KD = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (K-D = 123 nM). Deletions of Hup permease components hupD, hupG or hupDGC reduced Hn/Hb uptake, and complementation of Delta hupC and Delta hupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, DhupDGC did not completely eliminate [Fe-59]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall K-M of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (V-max = 23 pmol 10(9) cells(-1) min(-1)) to those of ferric siderophore transporters. In the Delta hupDGC strain uptake occurred at a threefold lower rate (V-max = 7 pmol 10(9) cells(-1) min(-1)). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e. g. Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, Delta hupDGC increased the LD50 of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.
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