Involvement of both the outer membrane heminhemoglobin receptor HmuR, and the lysine-specific gingipain, Kgp, in the acquisition of heme by Porphyromonas gingivalis
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The Gram-negative pathogen, Porhyromonas gingivalis,requires iron for growth. P gingivalis is capable of utilizing free heme, free iron, hemin, and hemoglobin as iron sources. In addition, hemoglobin bound to haptoglobin, and hemin complexed to hemopexin, can be used as heme sources, indicating that P. gingivalis must have a means to remove the hemin from these host iron-binding proteins. However, the specific mechanisms utilized by P. gingivalis for the extraction of heme from heme binding proteins and for iron transport are poorly understood. In this study, we report on the identification of a TonB-dependent hemoglobin/hemin receptor (HmuR) and demonstrate that both HmuR and the lysine-specific gingipain (Kgp) are involved in hemin/hemoglobin binding and utilization in P. gingivalis. HmuR shares amino acid homology with TonB-dependent outer membrane receptors of Gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin including HemR of Yersinia enterocolitica, ShuA of Shigella dysenteriae, HpuB of Neissria gonorrhoeae and N meningitidis, HmbR of N meningitidis, HgbA of Hbemophilus ducreyi, and HgpB of H influenzae. Southern blot analysis confirmed the presence of the hmuR gene and suggested genetic variability in the carboxy terminus of hmuR in P gingivitis strains 381, W50, and 53977. PCR amplification and subsequent enzymatic digestion of the entire hmuR gene from these P. gingivalis strains revealed that hmuR was conserved and suggested heterogeneity outside of the gene. We also identified directly upstream of the hmuR gene a gene which we designated hmuY. Upstream of the hmuY start codon, a region with homology to the Fur binding consensus sequence was identified. Fur is a global negative regulator of both genes involved in heme/iron transport and virulence determinants. RT-PCR analysis revealed that hmuR and hmuY were contrascribed and that transcription was negatively regulated by iron. Inactivation of hmuR resulted in a decreased ability of P. gingivalis to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sources. A P. gingivalis isogenic kgp mutant exhibited a decreased ability to bind hemoglobin. However, growth of this strain with hemoglobin was demonstrated to be similar to the parental strain. Inactivation of both hmuR and kgp genes resulted in a further decreased ability of P. gingivalis to bind hemoglobin, as well as a diminished ability to utilize hemin and hemoglobin as sole iron sources. Collectively, these results indicate that both HmuR and Kgp are required for the accumulation of hemin and hemoglobin in P. gingivalis.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (D.Sc.D.)--Boston University, Henry M. Golman School of Dental Medicine, 2001 (Oral Biology).Includes bibliographical references (leaves 99-128).
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