Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands

The Effects of Different Types of Antioxidants (Se, Vitamin E and Carotenoids) in Broiler Diets on the Growth Performance, Skin Pigmentation and Liver and Plasma Antioxidant Concentrations

Abstract This study investigated the effects of the addition of different antioxidants to broiler diets on their live performance, liver antioxidant composition and concentrations, immune response, and meat and skin color. A total of 945 three-day-old Ross 308 broiler chicks of both genders were randomly allocated to one of nine dietary treatments (n=105), with three replicates 35 chicks per pen, as follows: T1: control (commercially available corn-and soybean-based broiler diet); T2: selenium (control+0.5 mg/kg Sel-PlexTMSe yeast); T3: vitamin E (control+200 mg/kg Kavimix-E-50 a-tocopherol acetate); T4: lutein (control+100 mg/kg 5% Lutein Beads XB); T5: lycopene (control+100 mg/kg 5% Lyco Beads XB);T6: canthaxanthin (control+25 mg/kg 10% Carophyll(r)Red);T7: apo-ester (control+25 mg/kg 10% Carophyll(r)Yellow); T8: lutein+zeaxanthin (control+25 mg/kg Xamacol(r)); and T9: b-carotene (control+100 mg/kg 10% Rovimix(r)). Feed (starter, grower, developer and finisher phases) and water were provided ad libitum for 42 days. Body weights, feed intake, feed conversion values and plasma carotene concentrations were recorded weekly, and liver antioxidant concentrations were recorded at the end of the experiment. Newcastle disease (LaSota) vaccination was performed on day 22. HI titers were measured on days 14, 21, 35 and 42 to determine the effects of the antioxidants on the immune system. The addition of selenium, vitamin E, and carotenoid supplements to the commercial broiler diet significantly increased antioxidant accumulation in the liver and the plasma. All antioxidants assessed significantly improved the immune response. Selenium and vitamin E supplementation also significantly improved total carotenoid concentrations in the plasma. The carotenoids enhanced skin and meat color. None of the supplements tested influenced growth (p>0.05)