Fusobacterium Necrophorum

Factor H Binding as a Complement Evasion Mechanism for an Anaerobic Pathogen, Fusobacterium necrophorum

This information is current as Nathalie Friberg, Petteri Carlson, Erna Kentala, Petri S. of September 27, 2021. Mattila, Pentti Kuusela, Seppo Meri and Hanna Jarva J Immunol 2008; 181:8624-8632; ; doi: 10.4049/jimmunol.181.12.8624 http://www.jimmunol.org/content/181/12/8624 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Factor H Binding as a Complement Evasion Mechanism for an Anaerobic Pathogen, Fusobacterium necrophorum1

Nathalie Friberg,* Petteri Carlson,* Erna Kentala,† Petri S. Mattila,† Pentti Kuusela,*‡ Seppo Meri,*‡ and Hanna Jarva2*‡

Fusobacterium necrophorum subspecies funduliforme is an obligate anaerobic Gram-negative rod causing invasive infections such as the life-threatening Lemierre’s syndrome (sore throat, septicemia, jugular vein thrombosis, and disseminated infection). The aim of our study was to understand if and how F. necrophorum avoids C activation. We studied 12 F. necrophorum subsp. funduliforme strains isolated from patients with sepsis. All strains were resistant to serum killing after a 1-h incubation in 20% serum. The bacteria bound, at different levels, the C inhibitor factor H (fH). Binding was ionic and specific in nature and occurred via sites on both the N terminus and the C terminus of fH. Bound fH remained functionally active as a cofactor for factor I in the cleavage of C3b. Interestingly, patients with the most severe symptoms carried strains with the strongest ability to bind fH. An Downloaded from increased C3b deposition and membrane attack complex formation on the surface of a weakly fH-binding strain was observed and its survival in serum at 3.5 h was impaired. This strain had not caused a typical Lemierre’s syndrome. These data, and the fact that fH-binding correlated with the severity of disease, suggest that the binding of fH contributes to virulence and survival of F. necrophorum subsp. funduliforme in the human host. Our data show, for the first time, that an anaerobic bacterium is able to bind the C inhibitor fH to evade C attack. The Journal of Immunology, 2008, 181: 8624–8632. http://www.jimmunol.org/ usobacterium necrophorum is an obligate anaerobic criminate self from non-self cells (e.g., microbes), it plays a major Gram-negative rod. Two subspecies are identified: F. role in the opsonization and lysis of microbes. C activation prod- F necrophorum subsp. necrophorum (biovar A) involved in ucts can also attract and activate phagocytic cells to the area of diseases of animals, and F. necrophorum subsp. funduliforme (bio- microbial invasion. The C system can become activated directly by var B) involved in human pathology. F. necrophorum subsp. fun- the surface of pathogens via the alternative or the lectin pathway or duliforme is often viewed in the literature as part of the normal by specific Abs via the classical pathway. C activation results in oropharyngeal flora, but this point is still uncertain. F. necropho- the formation and deposition of C3b on microbe’s surface and rum causes local infections (e.g., tooth infections, acute sore throat formation of the lytic membrane attack complex (MAC)3. with tonsillitis and pharyngitis, persistent or chronic sore throat), To protect host cells from destruction or inappropriate activa- by guest on September 27, 2021 but also invasive infections such as Lemierre’s syndrome (1, 2). tion, C is inhibited by several specific proteins present in the fluid Lemierre’s syndrome is life-threatening and affects healthy ado- phase or on cell surfaces. Factor H (fH), a 150-kDa fluid phase lescents or young adults with a 2:1 male/female ratio (3). The protein, inhibits the alternative pathway (AP) by competing with syndrome is characterized by sore throat followed by jugular vein factor B for binding to C3b, accelerating the decay of C3 conver- thrombosis, septicemia, and distant infections with multiple ab- tase, and by acting as a cofactor for factor I-mediated degradation scesses (4, 5). Lemierre’s syndrome is rare and its incidence varies of C3b. from 0.8 to 1.5 cases per million per year (3, 6). In the preantibiotic Pathogenic microorganisms have developed several sophisti- era the mortality rate was 90%. Currently, mortality rates vary cated mechanisms to escape host defenses. To avoid C attack, from 4% to 18% (5, 7, 8). pathogens can inactivate C components or mimic C proteins (9). To cause disease, pathogens such as F. necrophorum must Furthermore, several pathogenic microbes have been shown to evade innate immune responses. Innate immunity, characterized bind C regulators such as fH or C4b binding protein (C4BP) from by rapid and non-specific responses, is the first-line of human de- blood plasma. Bacteria (e.g., Streptococcus pyogenes, Streptococ- fense against microorganisms. An important and efficient part of cus agalactiae, Streptococcus pneumoniae, Neisseria meningitidis, the innate immune response is the C system. As C is able to dis- Neisseria gonorrhoeae, Yersinia enterocolitica, Borrelia burgdorferi), some fungi such as Aspergillus fumigatus or Candida albi- cans, some viruses (e.g., HIV or NS1 filovirus), and also parasites *Department of Bacteriology and Immunology, Haartman Institute, University of such as Echinococcus spp. or Onchocerca spp. are able to recruit † Helsinki, Finland; Department of Otorhinolaryngology, Helsinki University Central and bind the C regulator fH (9). For S. pyogenes, S. pneumoniae, Hospital, Helsinki, Finland; and ‡HUSLAB, Helsinki University Central Hospital, Finland B. burgdorferi, N. meningitidis, and N. gonorrhoeae, fH binding Received for publication August 25, 2008. Accepted for publication October 20, has been shown to correlate with resistance to opsonophagocytosis 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 3 Abbreviations used in this paper: MAC, membrane attack complex; fH, factor H; with 18 U.S.C. Section 1734 solely to indicate this fact. AP, alternative pathway; C4BP, C4b-binding protein; FAA, fastidious anaerobic agar; 1 VBS, Veronal-buffered saline; NHS, normal human serum; FHR-1/-4, factor H-re- This study was supported by the Sigrid Juselius Foundation, the Academy of Fin- lated protein-1/-4; HIS, heat-inactivated serum; SCR, short consensus repeat; GVB, land, and the Helsinki University Central Hospital funds. gelatin Veronal buffer; FHL-1, factor H-like protein-1; MFI, mean fluorescence 2 Address correspondence and reprint requests to Dr. Hanna Jarva, Haartman Insti- intensity. tute, Department of Bacteriology and Immunology, P.O. Box 21, Fin-00014 Univer- sity of Helsinki, Helsinki, Finland. E-mail address: hanna.jarva@helsinki.fi Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 8625 or serum killing (10–14). For these bacteria, fH binding appears to GVB at 10,000 ϫ g to separate free protein from bacteria-bound proteins. confer protection against opsonization and/or MAC formation. After separation, activities in the pellet and in the supernatant were mea- Binding of fH to the microbial surface can thus be considered as an sured in a gamma counter. The ratio of bound to total radioactivities was calculated. In these assays S. pneumoniae PR 218 was used as positive important virulence factor. control (12). S. aureus was the negative control (S. Meri and H. Jarva, In this study we aimed to understand how F. necrophorum unpublished observation). avoids C activation. We observed that this anaerobic bacillus also The effect of ionic strength on the binding of 125I-fH to fusobacteria was binds human fH. The bound regulator remains functionally active studied by using increasing concentrations of NaCl in GVB (final concentrations of 150, 300, 450, and 600 mM). as a cofactor for factor I in the cleavage of C3b. The strains that In a competition assay, unlabeled fH was added to the reaction at the bound a higher amount of fH were more serum resistant than was same time as 125I-fH. The concentration of unlabeled fH varied between 1 a strain that bound less fH. Thus, our data show that binding of fH and 300 nM. BSA, at equimolar concentrations, was used as a negative contributes to the serum resistance of F. necrophorum. control.

Materials and Methods Elution of bound proteins Bacterial strains and culture conditions Bacteria were grown to mid-log phase, washed, and the concentration was ϫ 8 8 ϭ adjusted to 5 10 bacteria/ml in VBS. The cells (10 cells/assay) were The F. necrophorum strains (n 12) were clinical blood isolates from incubated in HIS (final concentrations of 0.1, 1, 2.5, 5 or 10%) or with patients with septicemia in the Helsinki University Central Hospital region. equimolar amounts of recombinant constructs of fH for 30 min at 37°C The strains were collected between 1994 and 2005. The strains were ini- with agitation. Bacteria were washed with VBS. After centrifugation, the tially identified at the species level by classical methods such as by colony supernatant of the last wash was saved. To elute surface-bound proteins, and Gram staining morphology as well as by growth characteristics. Fur- the pellet was resuspended in 50 ␮l of 0.1 M glycine-HCl (pH 2.5) and Downloaded from ther identification was done by real-time PCR according to the protocol incubated for 20 min at 37°C. Samples were centrifuged and the superna- previously described by Jensen et al. (15). This assay identified all the tants (eluates) were saved and neutralized with1MofTris-HCl (pH 9.5). strains as F. necrophorum subsp. funduliforme. Eluates and last washes were subjected to SDS-PAGE under nonreducing Strains were grown on fastidious anaerobic agar (FAA) for 24 h at 37°C conditions. The proteins were transferred to a nitrocellulose membrane. using AnaeroGen compact (Oxoid). Anaerobic BacT/ALERT SN culture Nonspecific binding sites were blocked with 5% milk-PBS and the mem- bottles (bioMe´rieux) were inoculated with the bacterial suspension and brane was incubated overnight at 4°C with a goat polyclonal anti-fH Ab bacteria were grown until mid-log phase. Bacteria were washed with Vero- (diluted 1/5000). After washing, the membrane was incubated for 1 h with http://www.jimmunol.org/ nal-buffered saline (VBS; 142 mM NaCl, 1.8 mM sodium barbital, 3.3 mM peroxidase-conjugated anti-goat IgG (diluted 1/5000). After washing, pro- barbituric acid (pH 7.4)) and the concentration was adjusted. teins were detected by ECL. The F. necrophorum strains incubated in VBS S. pneumoniae strain PR 218 was used as a positive control for direct instead of HIS were used as negative controls. fH-binding assays (12). It was grown in Todd-Hewitt broth with 0.2% yeast extract supplement to mid-log phase. S. aureus ATCC (American Type Culture Collection) 25923 was grown in Todd-Hewitt broth. Flow cytometry Proteins and Abs Bacteria were grown to mid-log phase, washed with VBS, and the concentration was adjusted to 5 ϫ 108 bacteria/ml in GVB. The cells (108 Pooled normal human serum (NHS) was obtained from healthy laboratory cells/assay) were incubated in HIS (final concentration 10%) for 30 min at personnel and stored at Ϫ70°C until used. Factor H-related protein-1 37°C with agitation. After washing with GVB, the bacteria were incubated

(FHR-1)-deficient serum was provided by a healthy donor. Heat-inacti- with a mAb against fH (196X) at the concentration of 5 ␮g/ml in 1% by guest on September 27, 2021 vated serum (HIS) was obtained by incubating NHS at 56°C for 30 min. BSA-VBS. We used an Alexa Fluor-488 conjugated anti-mouse IgG as fH, C3b, and factor I were purchased from Calbiochem. fH and C3b were second Ab (final concentration 1/200). After washing, the bacteria were radiolabeled with 125I using the iodogen method (16). Heparin was ob- resuspended in GVB and examined by flow cytometry (FACScan; BD tained from Sigma-Aldrich. Polyclonal goat anti-human fH was provided Biosciences). by Calbiochem. Mouse mAb 196X against fH has been described previously (17). Polyclonal rabbit anti-human C3c Ab was obtained from Dako. Cofactor assay for C3b inactivation Mouse Ab IgG against SC5b-9 was from Quidel. The peroxidase-conjugated donkey anti-goat IgG was provided by Jackson ImmunoResearch To analyze the functional activity of the bound fH, we used a C3b cofactor Laboratories. Alexa Fluor 488-conjugated donkey anti-rabbit and goat assay. Physiologically, fH is a cofactor for factor I in the cleavage of C3b. anti-mouse IgG Abs were from Invitrogen. Recombinant constructs of To study the cofactor activity of bacteria-bound fH, bacteria were incu- fH (short consensus repeat (SCR) 1–5, 1–6, 1–7, 8–11, 11–15, 15–20, bated with 0.1, 1, or 10% HIS. After washing, the pellet was resuspended and 8–20) were expressed using the baculovirus system and purified in VBS with 125I-C3b (100,000 cpm/sample) and factor I (final concentra- using Ni-Sepharose as described previously (18). Recombinant SCR5–7 tion 15 ␮g/ml). The incubation time was 1 h at 37°C. Samples were cen- and SCR19–20 constructs were expressed in Pichia pastoris as de- trifuged and the supernatants run into an SDS-PAGE gel under reducing scribed previously (19, 20). conditions to detect the cleavage of C3b. The gels were dried and subjected to autoradiography. A positive control, consisting of 125I-C3b (100,000 Serum resistance of the strains cpm/sample), factor I (15 ␮g/ml), and fH (10 ␮g/ml), was included in each series. 125I-C3b incubated without factor I and fH was used as a negative Freshly grown bacteria were washed three times in sterile VBS. The con- control. centrations were adjusted to 2 ϫ 106 bacteria/ml and 3 ϫ 105 bacteria (150 ␮l) were incubated in 20% or 75% NHS or Mg-EGTA serum at 37°C under anaerobic conditions with gentle agitation. Fifty-microliter portions of the C3b deposition suspensions were removed from the reaction tubes at 0, 1, 2, and 3.5 h time To study C3b deposition on bacterial surfaces, F. necrophorum strains points and diluted with VBS. To stop C activation, EDTA (final concen- were grown to mid-log phase and incubated with 50% NHS or Mg-EGTA- tration 10 mM) was added to the first dilution tube. After the last dilution, NHS (final concentration 5 mM MgCl , 10 mM EGTA) at 37°C with 100 ␮l of the solutions were plated on FAA plates and incubated for 48 h 2 agitation. The mixtures were incubated for 1, 2, 3, 4, 5, 15, and 30 min. at 37°C under anaerobic atmosphere. Bacteria incubated in 20% or 75% Bacteria were analyzed for C3b deposition with a rabbit anti-human C3c HIS were used as reference. Survival was counted as percentage of survival Ab and an Alexa Fluor 488-conjugated anti-rabbit IgG Ab was used as the of bacteria incubated with HIS at the corresponding time points. secondary Ab. Deposited C3b was detected by flow cytometry. Direct 125I-fH binding assays MAC formation The F. necrophorum, S. pneumoniae, and S. aureus strains were grown to mid-log phase. Bacteria were washed in VBS, resuspended in 0.1% gelatin MAC formation on fusobacterial membrane was studied by flow cytom- in VBS (GVB), and the concentration was adjusted to 2 ϫ 109 cells/ml. etry. The strains, grown to mid-log phase, were incubated in 50% normal Twenty microliters of bacteria (ϳ4 ϫ 107 cells/assay) was incubated with human serum at 37°C with agitation for 3, 5, 10, and 30 min. MAC was 20 ␮lof125I-fH (ϳ20,000 cpm/sample) for 30 min at 37°C with agitation. detected by a monoclonal anti-SC5b-9 Ab and Alexa Fluor-conjugated After incubation, the samples were centrifuged through 20% sucrose in anti-mouse IgG. 8626 Fusobacterium necrophorum BINDS FACTOR H

FIGURE 1. Binding of fH to F. necrophorum. A, Bacteria were incubated with 125I-fH and binding was assessed as percentage of offered protein. The results are shown as mean values Ϯ SD of ﬁve separate experiments with each performed in duplicate. S. pneumoniae PR 218 was used as a positive control and S. aureus ATCC 25923 as a negative control. The background represents radioactivity percentage in the pellet in the absence of bacteria. All F. necrophorum strains bound Ͼ10% of offered fH except strain 12 (5%). B, In a protein adsorption assay, F. necrophorum strains were incubated in 5% HIS. After elution, bound proteins were subjected to a Western blot analysis with a polyclonal anti-factor H Ab. Strains 4 and 5 bound fH and FHR-1 and FHR-4. Strain 12 bound clearly less fH than did the Downloaded from other strains, and it did not bind FHR-1 or FHR-4. Di- luted HIS (1/1000) was run for comparison. At this dilution, no FHR proteins could be detected. C, After incubation in 5% HIS deﬁcient in FHR-1, it appears that strain 5 bound strongly FHL-1 and fH, but strain 4 showed weaker FHL-1 binding. Strain 12 bound hardly any fH and no FHL-1. http://www.jimmunol.org/

of survival of bacteria incubated in the presence of HIS. At 1 h, the Results by guest on September 27, 2021 Description of the patients percentage of survival in 20% NHS ranged between 53% and Ͼ100% (data not shown). This result indicates that all strains are Clinical data were collected from 12 patients with a F. necropho- able to resist C attack and serum killing to various extents. rum infection. From each patient, age, sex, primary infection, and the presence of complications such as abscess, distant infections, 125 Direct binding of I-labeled fH to F. necrophorum and intravascular thrombosis were recorded. Except for one patient, aged 55 years, the ages of the patients ranged between 15 and Bacteria have developed several mechanisms to evade C activa- 28 years. Male/female ratio was 1:1. At admission to hospital, all tion. One of them is the binding of the AP inhibitor fH. Using a 125 patients presented with a F. necrophorum bacteremia. In all pa- binding assay with whole bacteria and I-fH, we observed that 11 tients, except patient 12, infection was initially located in the oro- fusobacteria out of 12 strains bound fH (Fig. 1A). Strains could be pharynx (dental infection, tonsillitis, or pharyngeal infection). Six arranged into three groups. In the first group, strains 1–9 bound patients (nos. 2, 6, 7, 9, 10, and 11) developed abscesses. In three factor H strongly (mean binding percentages varying from 20% to cases, jugular vein thrombosis was documented (nos. 3, 4, and 5), 42%). In the second group, represented by strains 10 and 11, the 125 and patient 6 presented with a disseminated intravascular coagu- binding percentages of I-fH were lower (mean values of 12% lation. Distant infection was observed in seven cases. The most and 16%, respectively). The third group consisted only of strain frequent location of the distant infections was the lungs (five pa- 12, which bound 5% of the offered protein. tients). Patient 5 developed a severe cardiorespiratory failure, and patient 6 died 7 days after his arrival to hospital due to multiorgan F. necrophorum binds fH from serum failure. To see if fH bound to fusobacteria directly from serum, F. necro- Patient 12 differed from the other cases. The final diagnosis for phorum strains were incubated with 0.1–10% serum and, after this 15-year-old adolescent was a facial skin cellulitis without any washing, bound proteins were eluted with 0.1 M glycine-HCl. The oropharyngeal infection, abscess, or intravascular complication. eluates were run into an SDS-PAGE gel. A representative experiment is shown in Fig. 1B. In all elution experiments, a large Ability of F. necrophorum strains to resist serum killing amount of fH was eluted from the surface of all strains except All strains were isolated from the bloods of the patients. The fact strain 12, to which a smaller amount of fH had become bound. For that all the strains had survived in human blood suggests that they the strongly fH-binding strains, it was still possible to elute bound had developed effective protection against immune responses. To fH after incubation in 0.1% of HIS (data not shown). study the ability of the bacteria to resist serum killing, the strains In these experiments, it could be seen that F. necrophorum was were incubated in 20% NHS or HIS for 1 h. After incubation, not only able to bind fH but also fH-related proteins-1 and -4 fusobacteria were plated on FAA plates and incubated under an- (FHR-1, FHR-4) (Fig. 1B). In this experiment, however, it was not aerobic conditions for 48 h. Survival was counted as a percentage possible to analyze binding of fH-like protein-1 (FHL-1) because The Journal of Immunology 8627

FIGURE 4. Mapping the fusobacterial binding site on fH. Recombinant constructs of fH SCR1–5, 1–6, 1–7, 8–11, 11–15, 15–20, and 8–20 (A) and 5–7 and 19–20 (B) were incubated with F. necrophorum strain 5. Bound proteins were eluted with glycine-HCl (pH 2.5) and detected by Western blotting using a polyclonal anti-fH Ab. F. necrophorum strain 5 bound to all other fragments than SCR8–11 or SCR11–15. This suggests two separate binding sites on fH: one at SCRs 5–7 and a second at SCRs 19–20.

125 Downloaded from FIGURE 2. Flow cytometry analysis of fH binding to fusobacteria. F. tion of I-fH binding, respectively. This result indicates that the necrophorum strains were incubated in 10% HIS. Bound fH was detected interaction between F. necrophorum and fH is ionic in nature. using a mAb (196X) and Alexa Fluor 488-conjugated secondary Ab. A representative experiment is shown with strains 12 (A), 10 (B),3(C), and Binding of fH to F. necrophorum is speciﬁc 5(D). For strain 12 no clear binding of fH was observed. The negative To study the speciﬁcity of the interaction between F. necrophorum controls included bacteria in GVB, bacteria incubated without HIS, and an and fH, a competition assay with unlabeled fH was employed.

experiment without the primary Ab. http://www.jimmunol.org/ Increasing concentrations of unlabeled fH (100 and 300 nM) inhibited binding of radiolabeled fH to F. necrophorum strain 5 by 36% and 70%, respectively (Fig. 3B). In the negative control, BSA of its comigration with FHR-1␤. Therefore, we used FHR-1-defi- was unable to inhibit 125I-fH binding. This result suggests that the cient HIS to detect binding of FHL-1. By incubating bacteria in interaction between F. necrophorum and fH is specific in nature. FHR-1-deficient HIS, we observed that F. necrophorum was able to bind FHL-1 (Fig. 1C). No FHL-1 binding was detected in F. necrophorum has two binding sites on fH strain 12. To further localize the sites on fH needed for binding to fusobac-

teria, recombinant fH constructs were used. Equimolar amounts by guest on September 27, 2021 Flow cytometry analysis of fH binding (0.33 ␮M) of recombinantly expressed SCR1–5, 1–6, 1–7, 8–11, To study if binding to the bacterial population was homogeneous, 11–15, 15–20, and 8–20 were incubated with strain 5, and bound bacteria were incubated in 10% HIS and analyzed by ﬂow cytom- proteins were eluted with an acid buffer. Eluates were separated by etry. Binding was detected by mAb 196X against fH and Alexa SDS-PAGE gel and analyzed by Western blotting using a poly- Fluor 488-labeled secondary Ab. clonal anti-fH Ab. With this method, no binding of SCR8–11 or The ﬂow cytometry assay results were in line with the direct SCR11–15 was seen, but SCR1–5, 1–6, 1–7, 8–20, and 15–20 binding and serum elution assays. All strains bound fH, but strain fragments were bound by F. necrophorum (Fig. 4A). These data, in 12 bound clearly less than the other strains. The representative addition to the results of the binding of FHL-1, FHR-1, and strains 3, 5, 10, and 12 are shown in Fig. 2. FHR-4, pointed to two distinct binding domains, one located in the

125 N-terminal part and the second in the C-terminal part of fH. The Binding of I-fH is ionic strength-dependent N-terminal binding site is located on SCR5–7 and the second site, To study the salt sensitivity of the fH-F. necrophorum interaction, in the C-terminal part of fH, is located between SCR15 and we used in a direct binding assay increasing concentrations (150– SCR20. To conﬁrm this result and further map the C-terminal site, 600 mM) of NaCl in the buffer. As shown in Fig. 3A, the binding F. necrophorum strain 5 was incubated with recombinant SCR5–7 of fH to F. necrophorum strain 5 decreased with increasing NaCl and SCR19–20. Elution and analysis by Western blotting con- concentrations. When NaCl concentration increased from 150 to ﬁrmed the presence of two binding sites: one in the N-terminal part 300, 450, and 600 mM, we obtained 41%, 62%, and 78% inhibi- (SCR5–7) and another in the C-terminal part (SCR19–20) region

FIGURE 3. Characterization of interaction between factor H and F. necrophorum. A, Inhibition of 125I-fH binding with NaCl. In a direct binding assay with strain 5, the increasing concentration of NaCl in the buffer (150, 300, 450, and 600 mM) decreased fH binding. This indi- cated an ionic interaction between F. necrophorum and fH. B, Competition assay with unlabeled fH. To demonstrate the speciﬁcity of the interaction between F. necrophorum and fH, unlabeled fH was added in the direct binding assay buffer (ﬁnal concentrations 10, 30, 100, and 300 nM). In- hibition of 125I-fH binding was obtained in the presence of 100 and 300 nM of unlabeled fH. 8628 Fusobacterium necrophorum BINDS FACTOR H

FIGURE 5. Bound fH is active as a cofactor for C3b inactivation. Three strains were tested: the weakly fH-binding strain 12, the intermediately fH-binding strain 10, and the strongly binding strain 5. Bacteria were incubated with HIS (ﬁnal concentrations 10%, 1%, or 0.1%) and, after washes, with factor I and 125I-C3b. The cleavage of C3b is visualized by the disappearance of the 110-kDa ␣Ј band and by the appearance of ␣Ј67-, ␣Ј43-, and ␣Ј41-kDa bands. For the strongly fH-binding strain 5, cleavage of C3b was observed after incubation in 10%, 1%, and 0.1% HIS. For the FIGURE 7. Binding of fH hinders the deposition of C3b on the surface Downloaded from weakly and intermediately binding strains, cleavage was seen only after of F. necrophorum. A, Strains 5 and 12 were incubated for 5 min in 50% 125 incubation in 10% HIS. A positive control, consisting of I-C3b, factor I, NHS. After washing, bacteria were stained with a polyclonal anti-C3c Ab 125 and fH, was included in each series. I-C3b incubated without factor I, and an Alexa Flur 488-labeled secondary Ab and analyzed by ﬂow cytom- and fH was used as a negative control. etry. Less C3b was deposited on the strongly fH-binding strain 5. B,A similar experiment was done in the presence of Mg-EGTA, which blocks the classical and lectin pathways. A higher amount of C3b was deposited of fH (Fig. 4B). Binding of the SCR15–18 region could not be on the weakly fH-binding strain surface. The results are shown as mean http://www.jimmunol.org/ excluded but was considered unlikely. values Ϯ SD of two separate experiments.

Fusobacteria-bound fH remains functionally active Factor H inhibits the AP of C by three different mechanisms: it accelerates the decay of AP convertase, it competes with factor B Protection of F. necrophorum against C attack for binding to C3b on self-cell surfaces, and cell-bound fH is also To test whether fH binding offered any survival advantage to F. acting as a cofactor for factor I-mediated degradation of C3b. In a necrophorum, we studied the kinetics of the bacterial survival in

cofactor assay, we studied the functional activity of F. necropho- NHS. Because all strains were resistant to killing after a 1-h in- by guest on September 27, 2021 rum-bound fH. The inactivation of C3b by factor I, in the presence cubation in 20% NHS and also because all strains bound fH, we of fH, was observed by the cleavage of the C3b ␣Ј-chain into increased the percentage of NHS to 75. We tested four F. necro- ␣Ј67-, ␣Ј43, and ␣Ј41-kDa fragments. Three strains were tested: phorum strains: 3 and 5 (strongly fH-binding strains), 10 (inter- the strongly fH-binding strain 5, the intermediately fH-binding mediately fH-binding strain), and 12 (weakly fH-binding strain). strain 10, and the weakly fH-binding strain 12 (Fig. 5). For strain Bacteria were incubated in 75% NHS or HIS. At 0, 1, 2, and 3.5 h, 5, the cleavage of 125I-labeled C3b was visible when bacteria were the bacteria were removed and cultivated on FAA plates for 48 h preincubated in 10%, 1%, or 0.1% HIS. For the intermediately and under anaerobic conditions. No clear difference was observed be- weakly fH-binding strains, the cleavage of C3b occurred only after tween the groups after 1 h (Fig. 6A). No clear difference was ob- incubation in 10% HIS. These results suggest that F. necropho- served between the strongly and intermediately fH-binding groups rum-bound fH remained functionally active as a cofactor for factor at any time point. However, strain 12 was the most sensitive to I and that the functional activity correlated with the amount of fH serum killing: the survival at 2 and 3.5 h was 27% and 11%, binding. respectively.

FIGURE 6. Binding of fH protects F. necrophorum against C attack. Four strains, representative of the three fH-binding categories (strongly binding strains 3 and 5, intermediately binding strain 10, and weakly binding strain 12), were tested for their serum resistance. Bacteria were incubated in 75% NHS. At each time point bacteria were removed and cultivated on FAA plates under anaerobic conditions. As a control, bacteria were incubated in HIS. The percentage of survival was calculated from the ratio between the number of CFU in NHS vs HIS. All strains were serum resistant at1hofincubation (A). However, after 1 h incubation, the weakly fH-binding strain 12 was more sensitive to C than were the other strains. In Mg-EGTA serum, strain 12 was less resistant than the strongly fH-binding strain 5 at 2 and 3.5 h incubation (B). The results are shown as mean values Ϯ SD of four separate experiments. The Journal of Immunology 8629

Mg-EGTA serum more C3b was deposited on the surface of strain 12 than on strain 5 (Fig. 7B). This suggests that bound fH on strain 5 inhibits C activation and protects the bacteria against C3b deposition.

Kinetics of MAC formation on the surface of F. necrophorum The formation of MAC on F. necrophorum surface was studied by flow cytometry. F. necrophorum strains 5 and 12 were incubated in NHS for 3, 5, 10, and 30 min at 37°C with agitation. After FIGURE 8. Kinetics of MAC formation on the surface of F. necropho- washes, they were stained with a mAb against C5b-9 neoepitope rum. Fusobacteria were incubated in 50% NHS for 3, 5, 10, and 30 min and stained by a monoclonal anti-SC5b-9 and Alexa Fluor 488-conjugated sec- and an Alexa Fluor 488 detection Ab. FACS analysis showed that ondary Ab. Flow cytometry shows at each time point a clear difference MAC formation on the surface of both F. necrophorum strains was between the strains 5 and 12. The strongly fH-binding strain 5 had depos- slower than C3b deposition. C3b deposition was complete in 5 ited less MAC than did the weakly fH-binding strain 12 to their surfaces. min, but MAC formation started at 5 min and the process still The results are shown as mean values Ϯ SD of two separate experiments. continued at 30 min (Fig. 8). The mean fluorescence intensity (MFI) observed for strain 5 at 30 min was similar to the MFI observed for strain 12 at 10 min. At each time point a clear dif- To eliminate the role of the classical pathway, we blocked it by Downloaded from ference between strains 5 and 12 was observed. Consistently, less using Mg-EGTA in the serum (final concentration 5 mM MgCl , 2 MAC was formed on strain 5. 10 mM EGTA). Under these conditions AP is the only active C pathway. Two strains (5 and 12) were tested (Fig. 6B). Both strains were more resistant to killing in Mg-EGTA serum than in NHS, Clinical correlation indicating that the classical pathway plays also a role in the killing The analysis of the clinical data and fH-binding tests pointed to a of F. necrophorum. However, strain 12 was less resistant than the http://www.jimmunol.org/ strongly fH-binding strain 5 to AP killing. At 3.5 h, only 34% of correlation between the severity of the disease and the ability of strain 12 survived vs 67% for strain 5. The correlation between the strains to bind the AP inhibitor. Interestingly, patients present- survival and the fH binding suggests that the binding of fH con- ing the most severe disease were carriers of strongly fH-binding tributes to the protection of F. necrophorum against C attack. strains (Table I). All patients who developed a distant infection or an intravascular complication carried a strongly fH-binding strain. Binding of fH prevents the deposition of C3b on the surface of Strains 5 and 6, involved in the most severe cases (cardiorespira- F. necrophorum tory failure, disseminated intravascular coagulation, and multior- To further investigate whether fH binding confers protection gan failure followed by death), bound also strongly to fH. against C activation and specifically against C3b deposition, we Compared with others, the weakly fH-binding strain 12 was by guest on September 27, 2021 compared the deposition of C3b between strains 5 and 12. After involved in a less severe disease. The patient had facial cellulitis. incubation in 50% NHS for 1, 2, 3, 4, 5, 10, and 30 min, the For all of the other strains, the port of entry was clearly the oro- fusobacteria were washed and stained with a polyclonal anti-C3c pharynx. However, for strain 12 no primary infection in the oro- Ab and an Alexa Fluor 488-conjugated secondary Ab. C3b depo- pharynx was reported. No complications, such as abscess, distant sition on the fusobacterial surfaces was fast and complete in 5 min infection, or intravascular thrombosis, were detected. This strain (data not shown). The weakly fH-binding strain 12 was more sus- was less resistant than the other strains in the serum killing assay. ceptible to C3b deposition than was the strongly fH-binding strain Thus, the decreased capacity to bind fH may contribute to the 5 (Fig. 7A). The difference was observed at each time point. In reduced serum resistance of strain 12 and a milder clinical picture.

Table I. Clinical characteristics of patients with F. necrophorum sepsisa

Strain Age Gender Primary Infection Complications Intravascular Thrombosis

Strong binding of fH 1 22 Female Acute tonsillitis Pneumonia, atelectasis None 2 16 Male Dental infection Back muscle abscess None 3 13 Female Acute tonsillitis Pneumonia Jugular vein 4 18 Male Pharyngeal infection Pneumonia, epigastric tenderness Jugular vein 5 18 Female Pharyngeal infection Pneumonia, severe cardiorespiratory failure Jugular vein 6 55 Male Dental infection Parapharyngeal abscess, lung empyema DICb 7 17 Male Acute tonsillitis Peritonsillar abscess None 8 21 Female Acute tonsillitis, history of None None chronic tonsillitis 9 15 Female Pharyngeal infection Lung abscess None Intermediate binding of fH 10 28 Male Acute tonsillitis Peritonsillar abscess None 11 18 Female Acute tonsillitis Peritonsillar abscess None Weak binding of fH 12 15 Male Facial cellulitis None None

a After collection of clinical data, each patient was considered by age, sex, primary infection, and presence of complications such as abscess, distant infections, and intravascular thrombosis. The strains are ranked into three groups: strong, intermediate, or weak binding of fH. The carriers of strongly fH-binding strains were more susceptible to develop a severe infection complicated by a distant infection. b Disseminated intravascular coagulation. Patient died 7 days after arrival to hospital due to multi-organ failure. 8630 Fusobacterium necrophorum BINDS FACTOR H

Discussion patient with a less severe disease (facial cellulitis). These data Fusobacteria are potentially invasive anaerobic bacteria that can suggest variability in the surface components between the strains cause severe infections. In this study we have shown that F. necro- and correlation of fH binding to virulence of the bacteria. phorum subsp. funduliforme is able to resist cytolytic C attack in Previously studied interactions between fH and bacteria point to serum and to bind the fluid phase C inhibitor fH. Strains that had one or more binding sites. Binding sites have been identified on the caused severe infections bound fH more strongly. Factor H-bind- N-terminal (SCRs 1–7), C-terminal (SCRs 15–20), or in the mid- ing occurred through two separate sites, one located on the N- dle part of fH (SCRs 8–15) (14, 26, 27). To localize the binding terminal part and another on the C terminus of fH. F. necropho- site for F. necrophorum on fH, we used three different assays. rum-bound fH remained active as a cofactor for factor I in the First, fH, FHR-1, and FHR-4 binding from serum was observed in cleavage of C3b, thereby promoting the survival of F. necropho- the fH-binding assay. By using FHR-1-deficient serum in the same rum in serum. Less C3b deposition and a delay in MAC assembly technique, binding of FHL-1 was also detected. Domains 1–5 of were observed on a strain that bound fH strongly (no. 5), compared FHR-1 are homologous with SCRs 6, 7, 18, 19, and 20 of fH and with a weakly binding strain (no. 12). those of FHR-4 with SCRs 6, 8, 9, 19, and 20 of fH (28). A new F. necrophorum subsp. necrophorum is an animal pathogen. It member of the fH protein family, called FHR-4A, was recently has been only rarely isolated from human beings (21). F. necro- described (29). It expresses the same homology with fH than does phorum subsp. funduliforme, related to both animal and human FHR-4 and possesses four more SCR domains than FHR-4, the infections, has been less studied than the subspecies necrophorum alternatively spliced product of the same gene. FHL-1 is identical (22, 23). In our study, all strains belonged to the subspecies fun- to the seven most N-terminal SCRs of fH and possesses four Downloaded from duliforme. For a long time, F. necrophorum has been considered as unique amino acids at the C terminus (30). The binding of FHL-1 part of the normal oropharyngeal flora. However, there is actually by F. necrophorum suggests the possibility of an N-terminal bind- no supporting data and this is under reevaluation (24). A recent ing site, whereas the binding of FHR-1 and FHR-4 suggests the study, using molecular biology tools for the detection of F. necro- possibility of a C-terminal binding site. Confirmation of these re- phorum, showed that fusobacteria could be part of normal flora sults was obtained by experiments with recombinant constructs of with a low carriage percentage in the control cohort (15). This fH. F. necrophorum was found to bind fH via two separate sites. raises the question of why certain fusobacterial strains become An N-terminal binding site is located within SCRs 5–7. No binding http://www.jimmunol.org/ pathogenic and cause invasive infections. was observed in the middle part (SCRs 8–15) of fH. Of the C- A strictly anaerobic pathogen usually needs special environmen- terminal constructs, F. necrophorum bound to SCR fragments tal conditions, such as tissue hypoxia caused by trauma, concom- 15–20 and 19–20. Thus, two separate binding sites on fH were itant infection, or neoplasia, to invade the host and cause disease. clearly identified for F. necrophorum: one at the N terminus (SCRs F. necrophorum seems to be different and can, on its own without 5–7) and a second one at the C terminus (SCRs 19–20). However, these environmental conditions, be pathogenic (25). It is a strictly we cannot totally exclude further binding sites on SCRs 1–4 and anaerobic primary pathogen causing local infections, such as acute SCRs 15–18. Factor H thus appears to bind to unknown receptor(s)

or chronic tonsillitis, but also life-threatening diseases, such as on F. necrophorum in a multipoint fashion. by guest on September 27, 2021 Lemierre’s syndrome. The invasive and severe diseases affect most Factor H plays an important role in the inactivation of deposited commonly healthy young adults. The mechanisms used by F. C3b. Factor I is the mediator of the reaction but it requires a co- necrophorum to escape innate immunity and to invade into tissues factor: fH, FHL-1, membrane cofactor protein (MCP; CD46), or C of young previously healthy adults are unknown. receptor type 1 (CR1; CD35). The site on fH for cofactor activity Complement evasion mechanisms of microorganisms have been for factor I-mediated degradation of C3b is located on the N ter- actively studied during the last decade. One well-known mecha- minus of fH on SCRs 1–4 (31). The C3b binding sites are located nism is the binding of fluid phase C inhibitors, fH and C4BP. The on SCRs 1–4 but also on SCRs 8–15 and 20 (17, 32). Our exper- AP of C is continuously activated at a low rate and a small amount iments showed that F. necrophorum-bound fH remained function- of C3b is constantly deposited on surfaces. The AP is also effi- ally active as a cofactor for factor I. This result indicates that even ciently recruited via activation of the classical pathway, for exam- if the F. necrophorum binding site at SCR5–7 is located near the ple, on the surfaces of microbes. Factor H down-regulates the AP active site, at the SCR1–4 region, bound fH can be still active. in a highly effective way. It acts as a cofactor for factor I-mediated When fusobacteria were incubated in NHS and in Mg-EGTA degradation of C3b, accelerates the decay of the AP C3 convertase, serum to focus on AP activation, less C3b was deposited on strain and competes with factor B by exhibiting a strong affinity for C3b 5 than on strain 12. This is in accordance with the fact that strain deposited on surfaces rich in polyanions. Thus, fH prevents the 5 exhibited a strong binding to fH and strain 12 bound fH only formation and decays the AP C3 convertase on self surfaces. weakly. In addition to its cofactor activity in the inactivation of Pathogenic microbes, on the other hand, express fH-binding mol- deposited C3b, fH competes with factor B for binding to C3b. Both ecules and thereby utilize fH binding as a C escape mechanism to mechanisms inhibit the formation of AP C3 convertase and the improve their survival in the host. amplification loop. The consequences are that less C3 is cleaved Several aerobic bacteria, parasites, and some fungi have been and thus less C3b is deposited, as observed for strain 5. described to bind fH (9). Little is known about the C escape mech- As fusobacteria were observed to survive in human serum, we anisms of anaerobic bacteria. In our experiments, most F. necro- also wanted to explore if the terminal C pathway and MAC for- phorum strains were able to bind fH to their surfaces. This result mation were affected by the binding of fH to F. necrophorum. was confirmed by three independent techniques using human se- When a strongly fH-binding strain (strain 5) was compared with a rum or purified fH protein. The level of fH binding by F. necro- weakly binding strain (strain 12), a delay in the kinetics of MAC phorum was in the same range as that by S. pneumoniae, which has formation on strain 5 was observed in the FACS assay. Because previously been shown in several studies to bind fH (12). How- other regulators (e.g., clusterin and vitronectin) are involved in the ever, there was variation in the strength of fH binding between regulation of the terminal pathway, we cannot connect these results strains. One of the strains (strain 12) differed from the other 11 exclusively to the binding of fH (33, 34). Inhibition of AP C3 strains by binding clearly less fH. This strain was isolated from a convertase generation would inhibit MAC formation, but further The Journal of Immunology 8631 work is needed to fully determine the role of fH binding in the Disclosures delay of MAC assembly. The authors have no financial conflicts of interest. All 12 F. necrophorum strains studied were isolated from blood. This shows their capacity to survive in an extremely inhospitable References environment. In our serum killing assay, all strains were serum 1. Batty, A., M. W. Wren, and M. Gal. 2005. Fusobacterium necrophorum as the resistant and had an excellent survival at 1 h. However, at 2 and cause of recurrent sore throat: comparison of isolates from persistent sore throat 3.5 h, the weakly fH-binding strain 12 died more readily than did syndrome and Lemierre’s disease. J. Infect. 51: 299–306. 2. Lemierre, A. 1936. On certain septicaemias due to anaerobic organisms. Lancet the other strains. 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