Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes

Iron, the second-most abundant metal in the Earth's crust, precipitates in water as an oxyhydroxide polymer, resulting in poor bio-availability (Neilands, 1974). Despite the paucity of soluble iron in solution, it is usually essential to pro- and eukaryotes from its activities as an oxygen carrier and metabolic or redox cofactor. Animals, plants and microbes compete for iron, and successful pathogens obtain it in the host environment. The majority of mammalian iron exists as haem (Hn) in myoglobin and haemoglobin (Hb), but in aerobic organisms iron functions in energy generation as an electron transfer chain cofactor, in lipid, steroid, xenobiotic and drug oxidation by cytochrome P450s, and in antioxidant defense mechanisms involving superoxide dismutases. Free Hn is toxic at excessive concentrations (Immenschuh et al., 2010, Reeder, 2010, Robinson et al., 2009) and bacteria regulate its accumulation (Stauff & Skaar, 2009). The upshot is that the multiple iron-containing systems of animals and bacteria lead to delicately balanced adversarial mechanisms for iron sequestration by the former (iron-binding proteins like ferritin, transferrin, lactoferrin, lipocalin), and iron acquisition by the latter organisms (siderophores and membrane iron/Hn transporters; Arslan et al., 2009, Braun, 2005, Konopka et al., 1982, Konopka et al., 1981, Singh et al., 2009, Tidmarsh et al., 1983). Bacteria produce membrane transport systems for Hn utilization during infections of animal hosts. In Gram-negative cells TonB-dependent outer membrane receptors, periplasmic binding proteins and ABC transporters capture Hn and concentrate it intracellularly (Perry et al., 2003, Schneider & Paoli, 2005, Wyckoff et al., 2004, Zhao et al., 2010). ATP hydrolysis energizes Hn transport through the inner membrane (IM) (Burkhard & Wilks, 2008, Stojiljkovic & Hantke, 1994, Thompson et al., 1999, Tong & Guo, 2007). In Gram-positive cells ABC transporters for Hn (Drazek et al., 2000, Skaar et al., 2004, Jin et al., 2005) underlie an extensive, loosely crosslinked peptidoglycan (PG) layer, that permits diffusion of small solutes through its 20–70 A diameter pores (Demchick & Koch, 1996, Meroueh et al., 2006, Touhami et al., 2004). Ferric siderophores like ferrioxamine B (FxB) and ferrichrome (Fc) need not interact with PG or proteins anchored to it: CM-resident ABC transporters are their only membrane uptake component (Jin et al., 2005). The different architecture of the Gram-positive bacterial cell wall makes the pathway of Hn uptake less certain, but sortase-anchored proteins in the PG matrix extract the porphyrin from iron-containing proteins like Hb by a cascade of binding reactions that ultimately deliver it to a CM transporter (Lei et al., 2003, Lei et al., 2002, Nygaard et al., 2006, Ran et al., 2010, Zhu et al., 2008). Hn/Hb transporters exist in Staphylococcus aureus (Grigg et al., 2010), Streptococcus pyogenes (Lei et al., 2002), Bacillis anthracis (Tarlovsky et al., 2010) and Listeria monocytogenes (Jin et al., 2005), that contribute to bacterial virulence (Braun, 2005, Carniel, 2001, Stork et al., 2004, Jin et al., 2005). In S. aureus the isd gene cluster encodes sortase B (SrtB), an SrtB-anchored Hn binding protein (IsdC), an ABC transporter and other sortase A-dependent PG-anchored proteins (Mazmanian et al., 2003). L. monocytogenes has a comparable srtB-containing genetic system, but deletion of its components, including the IsdC homolog Lmo2185, did not influence Hn/Hb uptake (Jin et al., 2005). Nor did Δlmo2185 or Δlmo2186 (Fig. 1) reduce the virulence of L. monocytogenes in a mouse model (Jin et al., 2005). Rather, iron acquisition from both the porphyrin and the protein required the activity of the putative ABC-transporter encoded by the hup chromosomal region (2498.3 – 2501.1 kb); ΔhupC impaired Hn and Hb uptake and decreased virulence (Jin et al., 2005). Figure 1 Chromosomal loci Because of uncertainties in the Gram-positive bacterial Hn transport pathway we further studied its uptake by L. monocytogenes. The putative Hn binding protein, HupD, displayed selectivity for the iron porphyrin, which contrasted the behavior of the ferric hydroxamate binding protein, FhuD, that showed broad recognition of numerous ferric siderophores. These data, and the impairment of Hn/Hb uptake by deletion of hupG (lmo2430), hupD (lmo2431)] or the whole operon (Δhup) reiterated the specificity of the Hup membrane permease. High sensitivity [59Fe]-Hn uptake assays defined the kinetic properties of the HupDGC transporter, identified a second Hn/Hb uptake system in L. monocytogenes, and showed a role for SrtB-anchored Lmo2185 in capture of the iron porphyrin. Sortase A -deficient bacteria, on the other hand, showed no defects in Hn/Hb acquisition.