The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Catherine A Butler,Yan Tan,Stuart G Dashper,Jacqueline E Heath,Michelle D Glew,Lianyi Zhang,Deanne V Catmull,Christine A Seers,Helen L Mitchell,Eric C Reynolds,Hasnah S G Khan,Benfang Lei

doi:10.1371/journal.pone.0111168

Abstract

Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Highlights

Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with specific bacteria in a biofilm and is a major cause of tooth loss [1]
Har contains the dual -C-X-XC- motifs involved in binding zinc in the S1 structural site which is found in some but not all Ferric Uptake Regulator (Fur) family members (Fig. 1)
A predicted heme regulatory motif (HRM) [39] -97C-98P-99L- which RlIrr uses for hemin binding (Fig. 1) was identified in the P. gingivalis Har sequence in place of the -H-X-H- S2 motif

Summary

Introduction

Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with specific bacteria in a biofilm and is a major cause of tooth loss [1]. Porphyromonas gingivalis is considered to be a principal pathogen in chronic periodontitis due to its close association with the disease in humans and its virulence in animal models [1,2,3,4,5]. P. gingivalis utilises manganese especially for protection from oxidative stress and intracellular survival in host cells [10,11]. P. gingivalis would be exposed to low iron/heme environments and oxidative stress during colonization and periods of disease quiescence. In response to this dynamic environment, P. gingivalis must tightly regulate iron homeostasis gene expression to survive. We have demonstrated the importance of ferrous iron uptake in P. gingivalis W50 using the ferrous iron transporter mutant W50FB1, which has half the iron content of the wild-type, and was avirulent in an animal model of disease [13]

Methods

Results

Conclusion