Iron acquisition in the dental pathogen Actinobacillus actinomycetemcomitans: what does it use as a source and how does it get this essential metal?

Abstract

Actinobacillus actinomycetemcomitans requires iron to grow under limiting conditions imposed by synthetic and natural chelators. Although none of the strains tested used hemoglobin, lactoferrin or transferrin, all of them used FeCl3 and hemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, hemoglobin, and hemin but not transferrin. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was sensitive to proteinase K treatment. A. actinomycetemcomitans harbors the Fur-regulated afeABCD locus coding for iron acquisition in isogenic and non-isogenic cell backgrounds. The genome of this oral pathogen also harbors several other predicted iron uptake genes including the hitABC locus, which restored iron acquisition in the E. coli 1017 ent mutant. However, the disruption of this locus in the parental strain did not affect iron acquisition as drastically as the inactivation of AfeABCD, suggesting that the latter system could be more involved in iron transport than the HitABC system. The genome of this oral pathogen also harbors an active copy of the exbBexbDtonB operon, which could provide the energy needed for hemin acquisition. However, inactivation of each coding region of this operon did not affect the hemin and iron acquisition phenotypes of isogenic derivatives. This observation suggests that the function of these proteins could be replaced by those coded for by tolQ, tolR and tolA as it was described for other bacterial transport systems. Interruption of a hasR homolog, an actively transcribed gene that is predicted to code for an outer membrane hemophore receptor protein, did not affect the ability of an isogenic derivative to bind and use hemin under chelated conditions. This result also indicates that A. actinomycetemcomitans could produce more than one outer membrane hemin receptor as it was described in other human pathogens. All strains tested formed biofilms on plastic under iron-rich and iron-chelated conditions. However, smooth strains attached poorly and formed weaker biofilms when compared with rough isolates. The incubation of rough cells in the presence of FeCl3 or hemin resulted in an increased number of smaller aggregates and microcolonies as compared to the fewer but larger aggregates formed when cells were grown in the presence of dipyridyl.