Porphyromonas endodontalis HmuY differentially participates in heme acquisition compared to the Porphyromonas gingivalis and Tannerella forsythia hemophore-like proteins

Introduction Porphyromonas gingivalis and Porphyromonas endodontalis belong to the Bacteroidota phylum. Both species inhabit the oral cavity and can be associated with periodontal diseases. To survive, they must uptake heme from the host as an iron and protoporphyrin IX source. Among the best-characterized heme acquisition systems identified in members of the Bacteroidota phylum is the P. gingivalis Hmu system, with a leading role played by the hemophore-like HmuY (HmuYPg) protein. Methods Theoretical analysis of selected HmuY proteins and spectrophotometric methods were employed to determine the heme-binding mode of the P. endodontalis HmuY homolog (HmuYPe) and its ability to sequester heme. Growth phenotype and gene expression analysis of P. endodontalis were employed to reveal the importance of the HmuYPe and Hmu system for this bacterium. Results Unlike in P. gingivalis, where HmuYPg uses two histidines for heme-iron coordination, other known HmuY homologs use two methionines in this process. P. endodontalis HmuYPe is the first characterized representative of the HmuY family that binds heme using a histidine-methionine pair. It allows HmuYPe to sequester heme directly from serum albumin and Tannerella forsythia HmuYTf, the HmuY homolog which uses two methionines for heme-iron coordination. In contrast to HmuYPg, which sequesters heme directly from methemoglobin, HmuYPe may bind heme only after the proteolytic digestion of hemoglobin. Conclusions We hypothesize that differences in components of the Hmu system and structure-based properties of HmuY proteins may evolved allowing different adaptations of Porphyromonas species to the changing host environment. This may add to the superior virulence potential of P. gingivalis over other members of the Bacteroidota phylum.


Figure S2.
Overexpression and purification of P. endodontalis HmuY Pe protein and its sitedirected mutagenesis variants.(A) HmuY Pe tagged with 6 histidines at the N terminus (6×His-HmuY Pe ) was overexpressed in E. coli after induction with IPTG and the bacterial cultures were collected before (IPTG-) and after (IPTG+) induction.(B) HmuY Pe was purified using cobaltimmobilized resin (TALON Superflow).Bacterial lysates (1) were centrifuged, 6×His-HmuY Pe protein was purified from the soluble fraction (2), and the efficiency of protein binding to the resin was verified by analyzing the unbound protein fraction (3).6×His-HmuY Pe protein was eluted from the resin using imidazole (4).To cleave off the 6×His tag, factor Xa was used (5).The protein was then incubated with nickel-immobilized resin (Ni-NTA), separating the purified HmuY Pe (6) from 6×His-HmuY Pe (7).(C) Site-directed mutagenesis variants of the HmuY Pe protein (M123A, H128A, H132A, and M163A) were overexpressed and purified using the same procedures.Proteins were separated by SDS-PAGE and stained with CBB G-250.Analysis of immunogenic epitopes and their cross-reactivity in denatured and native proteins.The reactivity of purified P. endodontalis HmuY Pe with anti-HmuY Pg (A) or anti-HmuY Tf (B) antibodies was examined using Western blotting and chemiluminescence staining.Proteins (100 ng) were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and visualized with Ponceau S staining.Purified HmuY Pg or HmuY Tf protein was examined as a control.HmuY Pe , HmuY Pg , and HmuY Tf are indicated with green, blue, and red arrows, respectively.Dot blotting analysis was performed using 20 and 100 ng of proteins (C).Antibodies from two immunized rabbits were used (#1 and #2).M, protein molecular mass marker.

Figure S3 .
Figure S3.Titration of the HmuY Pe protein with heme.5 µM protein was titrated with increasing concentration of heme under oxidizing (A) and reducing (B) conditions (the latter obtained by the addition of 10 mM sodium dithionite).Complex formation was monitored by UV-visible absorbance spectroscopy.The difference absorbance spectra were analyzed under oxidizing (C) and reducing (D) conditions.

Figure S4 .
Figure S4.Heme sequestration capacity of P. endodontalis HmuY Pe from hemoglobin.Heme transfer from methemoglobin (metHb) or reduced metHb (Hb red) was examined using PAGE, staining with TMB-H2O2, and subsequent visualization of proteins by CBB G-250 staining (A).P. gingivalis HmuY Pg was used as a control.Hemoglobin and HmuY Pe were incubated at equimolar concentrations under oxidizing (B) and reducing conditions (C), the latter formed by 10 mM sodium dithionite.Spectra were monitored using UV-visible absorbance spectroscopy.

Figure S5 .
Figure S5.Heme sequestration capacity of P. gingivalis HmuY Pg from human serum albumin (HSA).Heme transfer from HSA was examined using UV-visible absorbance spectroscopy.HSA and HmuY Pg were incubated at equimolar concentrations under oxidizing (A, C) and reducing conditions (B, D), the latter formed by 10 mM sodium dithionite.

Figure S6 .
Figure S6.P. endodontalis and P. gingivalis growth in culture media supplemented with various heme sources.Bacteria were cultured for two passages in a basal medium (BM) without adding a heme source (BM only).Then, bacteria were cultured in BM with the addition of heme, host hemoproteins (A, C), or selected HmuY proteins (B, C).Bacterial growth was monitored by measuring the optical density at 600 nm (OD 600 nm) over time and the results are shown as mean ± standard error.(C) Comparison of the bacterial growth rate depending on the heme source, determined by measuring the time required to reach an optical density at 600 nm of 0.25 by bacterial cultures, indicating the onset of the mid-log growth phase.Results are shown as the mean measured time (h) and the time range (min and max values).Hm, heme; Hb, human hemoglobin; HSA, human serum albumin; HmuY Pg , P. gingivalis HmuY; HmuY Pe , HmuY homolog from P. endodontalis; HmuY Tf , HmuY homolog from T. forsythia.

Figure
Figure S7.Analysis of immunogenic epitopes and their cross-reactivity in denatured and native proteins.The reactivity of purified P. endodontalis HmuY Pe with anti-HmuY Pg (A) or anti-HmuY Tf (B) antibodies was examined using Western blotting and chemiluminescence staining.Proteins (100 ng) were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and visualized with Ponceau S staining.Purified HmuY Pg or HmuY Tf protein was examined as a control.HmuY Pe , HmuY Pg , and HmuY Tf are indicated with green, blue, and red arrows, respectively.Dot blotting analysis was performed using 20 and 100 ng of proteins (C).Antibodies from two immunized rabbits were used (#1 and #2).M, protein molecular mass marker.