Hemophore-like proteins of the HmuY family in the oral and gut microbiome: unraveling the mystery of their evolution

Abstract

SUMMARY Heme (iron protoporphyrin IX, FePPIX) is the main source of iron and PPIX for host-associated pathogenic bacteria, including members of the Bacteroidota (formerly Bacteroidetes) phylum. Porphyromonas gingivalis , a keystone oral pathogen, uses a unique heme uptake (Hmu) system, comprising a hemophore-like protein, designated as the first member of the novel HmuY family. Compared to classical, secreted hemophores utilized by Gram-negative bacteria or near-iron transporter domain-based hemophores utilized by Gram-positive bacteria, the HmuY family comprises structurally similar proteins that have undergone diversification during evolution. The best characterized are P. gingivalis HmuY and its homologs from Tannerella forsythia (Tfo), Prevotella intermedia (PinO and PinA), Bacteroides vulgatus (Bvu), and Bacteroides fragilis (BfrA, BfrB, and BfrC). In contrast to the two histidine residues coordinating heme iron in P. gingivalis HmuY, Tfo, PinO, PinA, Bvu, and BfrA preferentially use two methionine residues. Interestingly, BfrB, despite conserved methionine residue, binds the PPIX ring without iron coordination. BfrC binds neither heme nor PPIX in keeping with the lack of conserved histidine or methionine residues used by other members of the HmuY family. HmuY competes for heme binding and heme sequestration from host hemoproteins with other members of the HmuY family to increase P. gingivalis competitiveness. The participation of HmuY in the host immune response confirms its relevance in relation to the survival of P. gingivalis and its ability to induce dysbiosis not only in the oral microbiome but also in the gut microbiome or other host niches, leading to local injuries and involvement in comorbidities.

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

“…Data obtained in our study showed that one of the P. gingivalis adaptive features can be assigned not only to better tolerance to oxygen and higher proteolytic activity but also to different properties of heme binding by HmuY Pg as compared to HmuY Pe . The strength of heme binding by HmuY proteins depends on the redox state of the external environment (Olczak et al, 2024) and based on our results is as follows: HmuY Pg >HmuY Pe >HmuY Tf . HmuY Pg , coordinating heme iron with two histidines, binds heme efficiently under both oxidizing and reducing conditions.…”

“…Analysis of the overall three-dimensional experimentally solved (P. gingivalis HmuY Pg and T. forsythia HmuY Tf ) or theoretically predicted (other selected Porphyromonas species) protein structures revealed that HmuY proteins identified in the Porphyromonas species are highly similar to the P. gingivalis HmuY Pg (Figure 1D), with the highest similarities observed in the core region (S ́miga et al, 2023a). As in so far characterized HmuY proteins (Olczak et al, 2024), the main differences in HmuY proteins identified in Porphyromonas species are visible in the structure of heme-binding pockets and differ mainly in the size of the entrance of the heme-binding pocket (Figure 1D). The data shown in Figure 1 allowed us to verify the spatial location of predicted amino acids involved in heme binding in HmuY homologs in analyzed Porphyromonas species.…”

“…As a control, we used HmuY Pg , which can directly sequester heme complexed with HSA (Figure 5A; Supplementary Figure S5) (Smalley et al, 2011;Sieminska et al, 2021), Hpx (Figure 5G) (Bielecki et al, 2018), and metHb (Supplementary Figure S4A) (Smalley et al, 2011). P. gingivalis can use a variety of heme sources thanks to the direct sequestration of heme from hemoproteins by HmuY Pg or its synergistic cooperation with highly active proteasesgingipains (Smalley and Olczak, 2017;S ́miga et al, 2023b;Olczak et al, 2024). Although P. endodontalis does not encode gingipain homologs, it produces proteases, albeit less active compared to P. gingivalis proteases (Figure 6A).…”

“…The Hmu system comprises six proteins (HmuYRSTUV) encoded on the P. gingivalis hmu operon. The first protein encoded on this operon, HmuY Pg , is the first representative of the novel HmuY family comprising hemophore-like proteins, different from classical hemophores or other hemophore-like proteins, such as P. gingivalis HusA protein ( Olczak et al., 2024 ). Although P. gingivalis can transport free heme directly using TonB-dependent outer-membrane receptor HmuR Pg , encoded downstream of the hmuY Pg gene, HmuY Pg protein facilitates this process through binding of heme and its delivery to HmuR Pg ( Smalley and Olczak, 2017 ).…”

“…Although P. gingivalis can transport free heme directly using TonB-dependent outer-membrane receptor HmuR Pg , encoded downstream of the hmuY Pg gene, HmuY Pg protein facilitates this process through binding of heme and its delivery to HmuR Pg ( Smalley and Olczak, 2017 ). Importantly, HmuY Pg can sequester heme directly from host hemoproteins or heme-binding proteins produced by cohabitating bacteria and deliver it to HmuR Pg ( Smalley and Olczak, 2017 ; Olczak et al., 2024 ). The functions of the other proteins encoded on the P. gingivalis hmu operon are unknown and they most likely play a role in heme transport into the bacterial cell and/or heme metabolism ( Smalley and Olczak, 2017 ; Rocha et al., 2019 ).…”