[PMC free article] [PubMed] [Google Scholar] 26. cofactor of numerous metabolic and enzymatic processes (10). Low solubility of the ferric iron at biological pH coupled with the sequestering of iron as a part of the innate immune system of the mammalian host restricts the availability of free iron to the invading microorganisms. Pathogenic bacteria, however, have adapted to this iron-restricted environment prevailing within the mammalian host and express unique iron acquisition systems (4). Siderophore-mediated iron uptake is commonly seen in several bacteria, while others, including and species, express specific outer SD 1008 membrane receptors that chelate the iron from host iron-containing molecules such as transferrin, lactoferrin, and heme compounds (27, 30). Since greater than 90% of the iron within the human body is associated with heme and heme-containing proteins, bacteria that can access these compounds and utilize the heme iron have a significant nutritional advantage. (15, 34), enterohemorrhagic O157:H7 (36), (25), (16), and (35) are some examples of bacterial pathogens that produce TonB-dependent outer membrane receptors that bind hemin, which is usually subsequently internalized with the help of ATP-binding cassette (ABC) transporters. A second type of heme uptake system, recognized in certain species such as (3) and (21), entails the secretion of heme-binding proteins called hemophores that bind heme and transport it to the cell surface to be internalized by specific cell surface receptors. In either of the systems, the hemin can either be internalized Ehk1-L as such or the iron alone can be internalized after it is released from your hemin at the SD 1008 cell surface (5). In addition, the association of iron with the expression of virulence factors is well known in several bacterial systems (11, 31, 33). Iron is an essential nutrient for pathogenic leptospires (9). Louvel et al. (23) performed random insertional mutagenesis with the saprophytic and recognized five hemin-requiring mutants. Three of these mutants experienced insertions in a gene encoding a protein that shares homology with the TonB-dependent ferric citrate receptor FecA of insertion into a in light of the data obtained from the whole-genome sequencing. Cullen et al. (7), in a detailed analysis of the outer membrane proteins of serovar Lai managed under different growth conditions, showed that LipL32, LipL36, pL50, and pL24 were influenced by SD 1008 both heat and iron. Efforts in our lab to understand iron acquisition in leptospires included the identification (LB191; GenBank accession number AE011607) and modeling of a putative TonB-dependent outer membrane receptor protein (32), which, despite showing low levels of similarity (39%) and identity (22%) with FepA of gene encoding the Fur regulator) and LB186 (encoding heme oxygenase) led us to hypothesize that this protein is an iron-regulated hemin-binding protein. We henceforth refer to this protein as HbpA (serovar Lai binds hemin and is expressed upon iron deprivation. In addition, we recognized another constitutively expressed hemin-binding protein with a molecular mass of approximately 44 kDa whose expression was impartial of iron levels. This protein, expressed by several leptospiral serovars, was found to be LipL41 by sequencing and immunoblotting with specific anti-LipL41 antibodies. MATERIALS AND METHODS Strains and growth conditions. The leptospiral serovars used in this study were obtained from the National Repository at the Regional Medical Research Centre, ICMR, Port Blair, Andaman and Nicobar Islands, India. The strains included DH5 (lab collection) and BL21(DE3)/pLysS (Novagen). Leptospires were managed in 0.2% agar-containing semisolid EMJH medium supplemented with 10% enrichment medium (Difco) at 30C. The cells were regularly produced in liquid EMJH medium (the concentration of iron was 10 g/ml) for about 10 days, and cells in the log phase were utilized for growth under high- and low-iron conditions (as detailed below). The strains were routinely produced in Luria-Bertani (LB) medium at 37C, with ampicillin (50 g/ml), kanamycin (50 g/ml), and chloramphenicol (34 g/ml) added as required for the appropriate strains. Chromosomal DNA isolation and Southern blot analysis. Chromosomal DNA was isolated according to standard protocols.