title: Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes.
abstract: 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 putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb) respectively. Deletion of lmo2185 in the srtB region reduced listerial [(59) Fe]-Hn transport, and purified Lmo2185 bound [(59) Fe]-Hn (K(D) = 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 [(59) Fe]-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 (K(D) = 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 ΔhupC and ΔhupG by chromosomal integration of hupC(+) and hupG(+) alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [(59) Fe]-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 ΔhupDGC 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, ΔhupDGC increased the LD(50) of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
authors.forename: Qiaobin
authors.initials: Q
authors.lastname: Xiao
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Moore
authors.forename: Yi
authors.initials: Y
authors.lastname: Shao
authors.forename: Hualiang
authors.initials: H
authors.lastname: Pi
authors.forename: Iharilalao
authors.initials: I
authors.lastname: Dubail
authors.forename: Alain
authors.initials: A
authors.lastname: Charbit
authors.forename: Salete M
authors.initials: SM
authors.lastname: Newton
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Klebba
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006418:Heme / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008089:Listeria monocytogenes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D008088:Listeriosis / Q000382:microbiology; D051379:Mice; D009876:Operon; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D006454:Hemoglobins; C509821:sortase B; D006418:Heme; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases