Genetic and molecular characterization of the iron acquisition systems of Actinobacillus actinomycetemcomitans

by Rhodes, Eric Robert.

Genetic and Molecular Characterization of the Iron Acquisition Systems of Actinobacillus actinomycetemcomitans by Eric Robert Rhodes Actinobacillus actinomycetemcomitans has been associated with a variety of human infections but mainly with Localized Aggressive Periodontitis. This pathogen grows under iron-limiting conditions imposed by synthetic and natural iron chelators. Utilization and binding assays showed that while all strains tested can bind lactoferrin, hemoglobin, and hemin but not transferrin, they can only use FeCl3 and hemin as iron sources. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was reduced when the cells were treated with proteinase K. Scanning electron microscopy showed that smooth strains form less complex biofilms when compared with rough isolates. The iron sources also affected biofilm formation in both rough and smooth isolates. The A. actinomycetemcomitans afeABCD iron transport system, expression of which is controlled by iron and Fur, as well as the hitABC iron transport system were identified. Transformation of the E. coli 1017 iron acquisition mutant with a plasmid harboring either afeABCD or hitABC promoted cell growth under iron-chelated conditions, which shows that the role of these systems are in iron acquisition. Insertions in each open reading frame showed that each one is needed to complement the growth defect of E. coli 1017 imposed by the iron-chelated conditions. A. actinomycetemcomitans isogenic mutants with interruptions in either afeA, afeB, afeC, afeD, hitA, hitB or hitC were all unable to grow under iron-chelated conditions in which the parental strain could grow. The afeABCD system mutants were more restricted in growth by iron-chelated conditions than the hitABC mutants. This suggests that the AfeABCD system has a bigger role in iron acquisition under the conditions tested. Mutations in exbB, exbD, tonB, components of the energy transducing system, and hasR, an outer membrane heme binding protein, did not affect the ability of this strain to use heme as the sole source of iron. This suggests the expression of alternative functions that play a role in heme acquisition. We also describe the implementation of 2-D PAGE and DNA microarray analysis to investigate global gene expression in response to different iron conditions. Preliminary results indicate differential gene expression in response to changes in the iron conditions. Genetic and Molecular Characterization of the Iron Acquisition Systems of Actinobacillus actinomycetemcomitans