Tannerella forsythia Tfo belongs to Porphyromonas gingivalis HmuY-like family of proteins but differs in heme-binding properties

Abstract

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other ‘red complex’ members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code an HmuY homolog. T. forsythia protein, termed as Tfo, binds heme, but preferentially in the ferrous form, and sequesters heme from the albumin–heme complex under reducing conditions. In agreement with that, the 3D structure of Tfo differs from that of HmuY in the folding of heme-binding pocket, containing two methionine residues instead of two histidine residues coordinating heme in HmuY. Heme binding to apo-HmuY is accompanied by movement of the loop carrying the His166 residue, closing the heme-binding pocket. Molecular dynamics simulations (MD) demonstrated that this conformational change also occurs in Tfo. In conclusion, our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme-binding properties.

Hc-hrg-2, a glutathione transferase gene, regulates heme homeostasis in the blood-feeding parasitic nematode Haemonchus contortus

“…7c.). The K d value was 7.538 ± 0.6023 μM (mean ± standard error, SE) which was in good agreement with previously published data on a heme binding protein Tfo of Tannerella forsythia [33]. These data indicate that rHc-HRG-2 binds to heme with a high affinity in vitro.…”

Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function

“…Escherichia coli Rosetta II (DE3) (Novagen) strain was grown under aerobic conditions in Terrific Broth (TB) with appropriate antibiotics as described previously (Bielecki et al, 2018).…”

“…Briefly, 70 μg of PgFur protein in a complex with MBP (for determination of structural zinc in PgFur variants with cysteine residues replaced by an alanine residue) or proteins in a complex with MBP, but digested with Xa factor (experiments with PgFur, PgFurΔ13aa, and EcFur) were incubated for 5 min at room temperature with Laemmli loading buffer without reducing agent and 0.2% SDS. The protein was separated by SDS-PAGE as described previously (Bielecki et al, 2018). Then, the gel was incubated for 5 min in 500 μM 4-(2-pyridyl) resorcinol solution (PAR) (Sigma Aldrich) in 25 mM HEPES, pH 8.0, containing 100 mM NaCl and 10% glycerol, followed by incubation in the presence of 50 mM H 2 O 2 .…”

PgRsp Is a Novel Redox-Sensing Transcription Regulator Essential for Porphyromonas gingivalis Virulence

“…Hemin chloride (ICN Biomedicals, Costa Mesa, CA, USA) solution was freshly prepared and protein-heme complexes were formed as described previously [20]. UV-visible spectra were monitored using a double beam Jasco V-750 spectrophotometer (10 mm path length).…”

“…Together with the heme-uptake Hmu system, gingipains engage in heme acquisition, which is essential for P. gingivalis survival due to the lack of ability to Microorganisms 2019, 7, 623 2 of 23 synthesize protoporphyrin IX [12]. Other virulence factors produced by P. gingivalis are hemagglutinins and fimbriae, which are responsible for interaction with host cells, as well as with other bacteria, enabling efficient P. gingivalis colonization of the oral cavity [18][19][20][21][22].…”