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Staphylococcus aureusis a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Intracellularity is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxicS. aureusstrains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death by intracellularS. aureusafter translocation into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication ofS. aureusin epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. Our study suggests that staphopain A is utilized byS. aureusto mediate escape from the host cell and thus contributes to tissue destruction and dissemination of infection.Author SummaryStaphylococcus aureusis a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. AlthoughS. aureusis not regarded as a professional intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle ofS. aureusremain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death mediated by intracellularS. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

Background Porphyromonas gingivalis possess the ability to invade host cells which prevents this pathogen from eradication by conventional periodontal therapy. Recently, antimicrobial photodynamic therapy (aPDT) was introduced to periodontal treatment as a complementary antibacterial method. The aim of this study was to evaluate the effect of toluidine blue-O (TBO) mediated aPDT on the viability of P. gingivalis invading gingival fibroblasts and keratinocytes in an in vitro model of infection. Methods Primary human gingival fibroblasts (PHGF) and telomerase immortalized gingival keratinocytes (TIGK) were infected with Pg ATCC 33277. Two concentrations of TBO (0.01 mg/mL, TBO-c1 and 0.001 mg/mL, TBO-c2) and a non-laser red light source (λ = 630 nm) were applied to treat both cell-adherent/intracellular Pg (the adhesion/invasion model) or exclusively the intracellular bacteria (the intracellular infection model). Results The median viability of cell-adherent/intracellular Pg in infected keratinocytes declined from 1.88 × 105 cfu/mL in infected cells treated with TBO without irradiation to 40 cfu/mL upon irradiation for 10 s with TBO-c1. At higher light doses a complete photokilling of P. gingivalis was observed. Pg from exclusively intracellular infection model was also efficiently eradicated as the residual viability dropped from 1.44 × 105 cfu/mL in control samples to 160, 20 and 10 cfu/mL upon irradiation for 10, 20 and 30 s, respectively. In the infected fibroblasts irradiation significantly reduced bacterial viability but did not completely eradicate the intracellular pathogen. Conclusions Antimicrobial PDT is effective in reducing the viability of intracellular periopathogens, however those residing within gingival fibroblasts seems to attenuate the photokilling effectiveness of this method.

Periodontal inflammation is one of the most common chronic inflammatory conditions in humans. Despite recent advances in identifying and characterizing oral microbiota dysbiosis in the pathogenesis of gum disease, just how host factors maintain a healthy homeostatic oral microbial community or prevent the development of a pathogenic oral microbiota remains poorly understood. An important determinant of microbiota fate is local antimicrobial proteins. Here, we report that chemoattractant protein chemerin, which we recently identified as a potent endogenous antimicrobial agent in body barriers such as the skin, is present in the oral cavity under homeostatic and inflammatory conditions. Chemerin and a chemerin-derived antimicrobial peptide are bactericidal against select bacteria strategically positioned in dental biofilm. Gingival crevicular samples from patients with gingivitis but not periodontitis contain abundant bioactive chemerin capable of inducing CMKLR1-dependent leukocyte migration. Gingipains secreted by the periodontopathogen P. gingivalis inactivate chemerin. Together, these data suggest that as an antimicrobial agent and leukocyte chemoattractant, chemerin likely contributes to antimicrobial immune defense in the oral cavity.

Background and Objectives Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. Material and Methods GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. Results Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B2 receptors (B2R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. Studies in TLR2−/−, B2R−/−, and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation of the TLR2→PMN→B2R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B2R-dependent generation of IFN-γ-producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B2R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis.

Background and Objective: Porphyromonas gingivalis, an anaerobic bacterium associated with adult periodontal disease, employs a number of pathogenic mechanisms, including protease/adhesin complexes (gingipains), fimbriae and hemagglutinins, to maintain attachment within colonized hosts. Here we examined the binding of gingipains and whole, live P. gingivalis cells to immobilized extracellular matrix proteins in the presence of soluble forms of the same proteins, to investigate whether this may constitute a colonization mechanism in the oral environment. Material and Methods: Binding of purified gingipain molecules and whole bacterial cells to immobilized matrix proteins was examined in the presence and absence of soluble competitors using enzyme-linked immunosorbent assays. Results: Purified gingipains or whole, live bacteria preferentially bound immobilized forms of matrix proteins, even in the presence of soluble forms of the same proteins. Fimbriae appeared to be redundant for adhesion to immobilized proteins in the presence of the gingipains, indicating that the protease/adhesins and hemagglutinins may be more important for adhesion under these conditions. Conclusion: The data presented here provide evidence for a model of adhesion for P. gingivalis within the fluid environment of the oral cavity, where preferential binding of matrix-located proteins over soluble forms facilitates colonization of the host.

PorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases, the gingipains, from the periodontal pathogenPorphyromonas gingivalis. It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel, membrane-traversingβ-strands. In this report, the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6×His) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly, those PorT mutations adversely affecting gingipain secretion enhanced expression of theporTgene but at the same time suppressed the transcription of the gingipainrgpBgene. Further, PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of theBacteroidetesphylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins.

Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease), which has sequence similarity to cysteine peptidases of the papain and calpain families. In this study, we biochemically characterize Tpr. We found that the 55 kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48 kDa, 37 kDa, and 33 kDa via sequential truncations at the N-terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane, where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations greater than 1 mM, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4 and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

The Porphyromonas gingivalis lysine-specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro-fragment, a catalytic domain with a C-terminal IgG-like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non-catalytic domains in pro-Kgp processing and maturation in P. gingivalis, the wild-type form of the gene was replaced with deletion variants encoding C-terminally truncated proteins, including KgpDeltaHA3/4 (Delta1292-1732 aa), KgpDeltaHA2-4 (Delta1157-1732 aa), KgpDeltaHA1-4 (Delta738-1732 aa), KgpDeltaC-term/HA (Delta681-1732 aa) and KgpDeltaIg/C-term/HA (602-1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpDeltaC-term/HA and kgpDeltaIg/C-term/HA transcripts, no Kgp-specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp-specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane-associated and -secreted forms. While both gingipain K activity and the protein were cell membrane-associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C-terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin-like subdomain is indispensable for proper folding and expression of the gingipains.

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis., Author Summary Clinical and epidemiological data indicates that chronic periodontal disease (PD), one of the most prevalent infectious inflammatory disease of mankind, is linked to systemic inflammatory diseases such as cardiovascular diseases (CVD), rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD). Nevertheless, the causative mechanisms of association between PD and chronic inflammatory diseases are very poorly understood. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic response to citrullinated epitopes. In present study we show that infection with viable periodontal pathogen Porphyromonas gingivalis but not another oral bacterium (Prevotella intermedia), exacerbated CIA, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique enzyme peptidylarginine deiminase, which converts arginine residues in proteins to citrulline. This knowledge may create new perspectives in the treatment and prevention of RA in susceptible individuals.

Porphyromonas gingivalis is a Gram-negative bacterium associated with the development of periodontitis. The evolutionary success of this pathogen results directly from the presence of numerous virulence factors, including peptidylarginine deiminase (PPAD), an enzyme that converts arginine to citrulline in proteins and peptides. Such posttranslational modification is thought to affect the function of many different signaling molecules. Taking into account the importance of tissue remodeling and repair mechanisms for periodontal homeostasis, which are orchestrated by ligands of the epidermal growth factor receptor (EGFR), we investigated the ability of PPAD to distort cross talk between the epithelium and the epidermal growth factor (EGF) signaling pathway. We found that EGF preincubation with purified recombinant PPAD, or a wild-type strain of P. gingivalis , but not with a PPAD-deficient isogenic mutant, efficiently hindered the ability of the growth factor to stimulate epidermal cell proliferation and migration. In addition, PPAD abrogated EGFR-EGF interaction-dependent stimulation of expression of suppressor of cytokine signaling 3 and interferon regulatory factor 1. Biochemical analysis clearly showed that the PPAD-exerted effects on EGF activities were solely due to deimination of the C-terminal arginine. Interestingly, citrullination of two internal Arg residues with human endogenous peptidylarginine deiminases did not alter EFG function, arguing that the C-terminal arginine is essential for EGF biological activity. Cumulatively, these data suggest that the PPAD-activity-abrogating EGF function in gingival pockets may at least partially contribute to tissue damage and delayed healing within P. gingivalis -infected periodontia.

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.

Enhanced production of proinflammatory bradykinin-related peptides, the kinins, has been suggested to contribute to the pathogenesis of periodontitis, a common inflammatory disease of human gingival tissues. In this report, we describe a plausible mechanism of activation of the kinin-generating system, also known as the contact system or kininogen-kallikrein-kinin system, by the adsorption of its plasma-derived components such as high-molecular-mass kininogen (HK), prekallikrein (PK), and Hageman factor (FXII) to the cell surface of periodontal pathogen Porphyromonas gingivalis . The adsorption characteristics of mutant strains deficient in selected proteins of the cell envelope suggested that the surface-associated cysteine proteinases, gingipains, bearing hemagglutinin/adhesin domains (RgpA and Kgp) serve as the major platforms for HK and FXII adhesion. These interactions were confirmed by direct binding tests using microplate-immobilized gingipains and biotinylated contact factors. Other bacterial cell surface components such as fimbriae and lipopolysaccharide were also found to contribute to the binding of contact factors, particularly PK. Analysis of kinin release in plasma upon contact with P. gingivalis showed that the bacterial surface-dependent mechanism is complementary to the previously described kinin generation system dependent on HK and PK proteolytic activation by the gingipains. We also found that several P. gingivalis clinical isolates differed in the relative significance of these two mechanisms of kinin production. Taken together, these data show the importance of this specific type of bacterial surface-host homeostatic system interaction in periodontal infections.

Porphyromonas gingivalis HmuY is a putative heme-binding lipoprotein associated with the outer membrane. It is part of an operon together with a gene encoding an outer-membrane hemin utilization receptor (HmuR) and four uncharacterized genes. A similar operon organization was found in Bacteroides fragilis and B. thetaiotaomicron, with the former containing an additional HmuY homologue encoded upstream of the hmuR-like gene. In P. gingivalis cultured under heme-limited conditions, a approximately 1-kb hmuY transcript was produced at high levels along with some approximately 3.5 and approximately 9-kb transcripts. Compared with the parental strain, mutants deficient in hmuY or hmuR or hmuY-hmuR gene function grew more slowly and bound lower amounts of hemin and hemoglobin. Significantly, they grew more slowly or were unable to grow when human serum was used as the sole iron/heme source. Analysis of the hmu promoter showed that it is regulated by iron. The HmuY protein normally occurs as a homodimer, but in the presence of hemin it may form tetramers. These results show that HmuY may be the first reported member of a new class of proteins in Porphyromonas and Bacteroides species involved in heme utilization, a function being exerted in conjunction with HmuR, an outer-membrane heme transporter.

Heme utilization inPorphyromonas gingivalisrequires the participation of an outer membrane hemin/hemoglobin receptor, HmuR, the lysine-specific gingipain proteinase (Kgp) and arginine-specific gingipain proteinase (Rgp). In this study, the expression ofhmuR,kgpandrgpAgenes in response to growth with different heme sources was examined by reverse transcription-polymerase chain reaction and enzyme-linked immunoassay. Coordinate regulation ofhmuR,kgpandrgpAgene expression was evaluated through utilization ofP. gingivalis hmuRandkgpmutants or by selective inactivation of proteinases with Kgp- and Rgp-specific inhibitors. We observed that expression of thekgpandrgpAgenes was not tightly regulated by heme, but rather by the growth phase. In contrast, expression of thehmuRgene was negatively regulated by heme, while growth ofP. gingivaliswith human serum resulted in increasedhmuRexpression. AP. gingivalis kgpisogenic mutant demonstrated significantly increasedhmuRgene expression, and inactivation of Kgp and Rgp activity by specific inhibitors up-regulatedhmuRgene transcription. Moreover, inactivation of Kgp up-regulatedrgpAtranscription. Finally, aP. gingivalis hmuRmutant exhibited repressedkgpgene expression and lysine-specific proteinase activity. Collectively, these results indicate thatkgp,rgpAandhmuRgene transcription is coordinately regulated and may facilitate greater efficiency of heme utilization inP. gingivalis.

Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the β-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth of P. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a useable source of heme for growth and may contribute to the proliferation of P. gingivalis within periodontal pockets in which erythrocytes are abundant.

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of hemebound HmuY reveals a new all-b fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection. © 2009 Wójtowicz et al., This study was supported by grants from Spanish ministries (BIO2006-02668, BFU2006-09593, BIO2009-XXXXX, PSE-010000-2007-1, and CONSOLIDER-INGENIO 2010 Project “La Factoría de Cristalización” (CSD2006-00015)) and by grant 2005SGR00280 from the Generalitat de Catalunya. Additional funding was obtained from grants N401 029 32/0742 and 1642/B/P01/2008/35 from the Department of Scientific Research, Polish Ministry of Science and Higher Education; from US National Institutes of Health grant DE 09761; and from the European Union through EU FP6 Strep Project LSHG-2006-018830 “CAMP” and EU FP7 Collaborative Project 223101 “AntiPathoGN”. Further funding was provided by ESRF (http://www.esrf.eu/) for data collection