Molecular characterization of Porphyromonas gingivalis heme utilization systems--role of HmuR and gingipains in heme utilization

Thesis (Ph.D.)--Boston University, Henry M. Goldman School of Dental Medicine.PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Porphyromonas gingivalis , a Gram-negative anaerobic pathogen of periodontal diseases, requires iron in the form of heme (a term used to denote either the ferrous or ferric form of iron protoporphyrin IX) for growth. P. gingivalis is capable of utilizing a broad range of heme-containing compounds such as hemoglobin, hemoglobin-bound haptoglobin, hemin-bound hemopexin and hemin-saturated serum. Heme and hemoglobin utilization in P. gingivalis requires the participation of an outer membrane protein HmuR (heme utilization receptor), as well as cysteine proteinase gingipains (Lysine-specific gingipain Kgp and Arginine specific gingipains Rgps). However, the specific mechanisms utilized for heme acquisition are poorly understood. In this study, the role of HmuR in heme utilization was characterized in both E. coli and P. gingivalis . Molecular interaction between HmuR and hemin/hemoproteins was also characterized by construction and analysis of HmuR site-directed mutants. Our results support the direct role of HmuR in heme utilization. Hemoprotein utilization in P. gingivalis requires the participation of HmuR conserved residues. The HmuR residues 95 and 434, as well as the NPDL motif, seem to be involved in whole cell binding of hemoproteins; while the YRAP motif does not. All these residues seem essential for serum hemoprotein utilization. Analyses of HmuR by homology modeling provided a structural basis for functional analysis and supported the results from mutagenesis studies. In addition, expression of the hmuR, kgp and rgpA genes in response to different heme sources was also examined. We found that expression of the hmuR gene was negatively regulated by heme, while expression of the kgp and rgpA genes seemed to be regulated by growth phase. These different regulatory mechanisms, as well as the coordinate expression between HmuR and gingipains, indicate a complementary regulation mechanism for optimal heme utilization in P. gingivalis.2031-01-0