Iron‐ and hemin‐dependent gene expression of Porphyromonas gingivalis

Abstract

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron-dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron- and hemin-dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole-genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron-replete and iron-depleted conditions as well as under hemin-replete and hemin-depleted conditions. Under iron-depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron-depleted conditions to those from hemin-depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron-dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron-regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron-stressed bacterium than the iron-replete P. gingivalis cells. The major iron-regulated proteins encoded by PG1019-20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole-genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.

Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

“…P. gingivalis requires iron for growth; a number of P. gingivalis proteins require iron to function (57). Both iron (57) and hemin (34, 57) levels affect gene expression in multiple biological processes (e.g., metabolism, adherence and invasion, virulence factors, and oxidative stress).…”

The interplay between iron, haem and manganese in Porphyromonas gingivalis

“…gingivalis W83 has one Fur orthologue (PG0465) encoded in its genome, but the molecular mechanisms of iron-dependent regulation appear to be novel in P. gingivalis as the deletion of the Fur protein had no effect on the expression of iron-regulated genes or manganese-regulated genes [67,134]. Instead, this Fur orthologue, called Har for haem associated regulator, was demonstrated to regulate haem-responsive biofilm formation [134].…”

“…The gene encoding HtrE is located adjacent to an operon encoding a putative ATP binding cassette transport system with sequence similarity to haem transport systems of other bacteria, thus together, these genes may encode a haem transport system [95]. The PG1019-1020 locus of P. gingivalis encodes a predicted outer membrane lipoprotein and an outer membrane TonB-linked receptor respectively that are greatly increased in abundance during haem-limitation [62] and iron-limitation [67], also suggesting a role in haem/iron transporter (Fig. 2).…”

“…Due to the large amount of iron required by this complex, genes encoding Rnf proteins are down-regulated when P. gingivalis is grown in iron-limited conditions [67].…”

“…Instead Har conferred the ability to respond to environmental haem and develop biofilms, both of which are key attributes for the in vivo survival and pathogenicity of P. gingivalis. P. gingivalis is an iron-dependent Gram-negative bacterium that has a distinct iron-responsive regulon [67] but does not utilise a member of the Fur superfamily to regulate iron homoeostasis, instead linking the transport of haem and ferrous iron from exogenous sources with quorum sensing via LuxS. James et al [137] have shown that LuxS was required for a 1.5-fold increase in transcript levels of the ferrous ion transport system but negative regulators of this system have not yet been identified.…”