FACIN, a Double-Edged Sword of the Emerging Periodontal Pathogen Filifactor alocis: A Metabolic Enzyme Moonlighting as a Complement Inhibitor

Abstract

Periodontal disease is one of the most common inflammatory infectious diseases worldwide and it is associated with other syndromes, such as cardiovascular disease or rheumatoid arthritis. Recent advances in sequencing allowed for identification of novel periodontopathogens such as Gram-positive Filifactor alocis, but its virulence mechanisms remain largely unknown. We confirmed that F. alocis is a prevalent species in periodontitis patients, and we also observed strong correlation of this bacterium with clinical parameters, highlighting its role in the pathogenesis of the disease. Further, we found that preincubation of human serum with F. alocis resulted in abolished bactericidal activity and that F. alocis was surviving readily in full blood. We demonstrated that one of the key contributors to F. alocis complement resistance is a unique protein, FACIN (F. alocis complement inhibitor), which binds to C3, resulting in suppression of all complement pathways. Interestingly, FACIN is a nonclassical cell surface protein, a cytosolic enzyme acetylornithine transaminase, for which we now identified a moonlighting function. FACIN binds to C3 alone, but more importantly it also captures activated complement factor 3 within the complex with factor B, thereby locking in the convertase in an inactive state. Because of the indispensable role of alternative pathway convertase in amplifying complement cascades, its inhibition by FACIN results in a very potent downregulation of activated complement factor 3 opsonization on the pathogen surface, accompanied by reduction of downstream C5 cleavage.

Fig. 1. F. alocis produces several virulence factors affecting bactericidal activity of human serum and resists phagocytosis in blood

A) F. alocis ATCC 35896 or B) P. gingivalis were incubated for 30 min in anaerobic conditions with PBS (untreated), pronase or 0.02% NaN₃. Heat-killed F. alocis and P. gingivalis were prepared by incubation for 10 min at 72°C. After treatment, F. alocis or P. gingivalis (5 × 10⁸ CFU) were washed once in GVB⁺⁺ and incubated with 0.5%(A)/1%(B) NHS or ΔNHS for 60 min at 37°C in anaerobic conditions. Next, 100 μL of E. coli (in (A) 10⁶ CFU or (B) 10⁵ CFU) were added to all the samples and incubated further for 30 min at 37°C. Finally, aliquots were removed, diluted serially, spread onto LB agar plates and incubated at 37°C. E. coli colonies were counted and the numbers of surviving bacteria were calculated. No colonies of F. alocis were formed on LB under these aerobic conditions. The dashed line divides samples that were tested in separate experiments. C) Bacteria were incubated for 30 and 60 min at 37°C with freshly collected human blood. After incubation, aliquots were removed, serially diluted, and plated on appropriate agar plates. Survival was calculated as percentage of survival compared to the inoculum. In (A–B) statistical significance of observed differences was estimated compared to untreated F. alocis or P. gingivalis, respectively; in (C) - between commensal V. parvula and other species. The significance was estimated using two-way analysis of variance and Bonferroni post-test; **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, if nothing indicated – non-significant.

Fig. 2. F. alocis is recognized by all complement pathways leading to C3b deposition and also binds directly C3

A–C) F. alocis was incubated for 1 h with increasing concentrations of NHS or heat inactivated NHS (ΔNHS) diluted in GVB⁺⁺ (A) or Mg-EGTA (B). Similarly, (C) F. alocis or (D) zymosan particles were incubated with NHS, C1q-, C2-, FB-deficient serum and ΔNHS diluted in GVB⁺⁺, or with NHS, ΔNHS and FB-, and C1q-deficient serum diluted in Mg-EGTA. E) F. alocis grown in a planktonic or a biofilm culture was incubated for 1h with NHS and ΔNHS in GVB⁺⁺. F) M. catarrhalis RH4 and its isogenic mutant lacking UspA1/A2 were incubated with increasing concentrations of NHS and ΔNHS in Mg-EGTA. The dashed line separates results obtained for the two strains. In A–F deposited C3b/bound C3 was detected on bacterial surface with specific polyclonal antibodies using flow cytometry. Deposition/binding of C3b/C3 is shown as geometric mean fluorescence intensity (GMFI). In E the data were normalized to the highest GMFI obtained for the sample containing planktonic bacteria in 15% NHS. Means of three independent experiments are presented with bars indicating standard deviation (SD). Either standard (C, D, E) or matched (A, B, F) two-way analysis of variance ANOVA (yielding in all cases p < 0.0001) and Tukey or Bonferroni post-test were used to calculate significance; **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, if nothing indicated – non-significant. In (A, B, E) the statistical significance of differences is shown compared to Ctrl (control – bacteria without serum, incubated with detection antibodies), in (C, D) – compared to NHS, in (E) - between two culture forms. We observed deposition of C3b via both CP and AP with higher degree of deposition on the planktonic form compared to bacteria grown in biofilm. Furthermore, C3 was also absorbed from ΔNHS in a complement independent manner compared to what was previously observed for M. catarrhalis.

Fig. 3. F. alocis binds directly C3 via ionic interactions and prevents C3 convertase formation

A) Processing of C3 during complement activation. C3 convertases cleave C3 (composed of α-chain and β-chain) to C3b and the anaphylatoxin C3a. The thioester of C3b interacts covalently with molecules in its vicinity such as microbial surfaces. C3b is then inactivated by factor I in combination with various cofactors in several steps. First, small fragment C3f is released from the middle part of α-chain resulting in the formation of inactive iC3b, the major opsonin (not shown). Further cleavages liberate the large C3c fragment and leave the smaller C3dg fragment attached to microbial surface. Subsequently C3dg fragment is truncated to yield stable C3d. C3 treated with methylamine (C3met) corresponds conformational and functionally to C3b but still has C3a attached. B) F. alocis and M. catarrhalis were incubated for 1 h with intact C3, C3-met or various C3-fragments (100 or 25 μg/mL respectively) (C) F. alocis was incubated for 1 h with C3-met in the presence of increasing concentrations of NaCl. (B–C) bound proteins were detected on bacterial surface with specific antibodies anti-C3c or anti-C3d using flow cytometry. We observed that F. alocis binds intact C3 as well as its fragments C3b/C3met, C3c and C3d (B) and that this interaction is sensitive to ionic strength (C). Means of three independent experiments are presented with bars indicating SD. Statistical significance of observed differences compared to (B) corresponding Ctrl (control – bacteria without any protein, incubated with detection antibodies) or (C) physiological NaCl (0.15 M) was estimated using one-way analysis of variance ANOVA (yielding p < 0.0001) and a Dunnett post-test; *** p<0.001, ** p<0.01, * p<0.05, if nothing indicated – non-significant. (D–E) F. alocis was incubated for 1 h with C3 to allow binding of the protein to the bacterium (Step 1), washed, and subsequently FB and FD were added to the samples, which were incubated further for another hour (Step 2). As a control, all components of the alternative pathways (C3, FB and FD) were added to the bacteria at the same time (Step 2). Next, bacteria were washed and suspended in reducing sample buffer. Proteins were separated by SDS-PAGE and blotted onto PVDF membrane. (D) Conversion of FB to Bb (D) and cleavage of α-chain of C3 (E), both indicative of active alternative pathway convertase formation on bacterial surface were detected with specific pAbs. When C3 was added to the bacteria simultaneously with FB and FD, we observed its conversion to C3b as well as activation of FB. However, C3 preincubated first with bacteria lost this ability.

Fig. 4. Several of cell surface-attached proteins of F. alocis bind C3

Surface proteins of F. alocis were isolated and separated using 2D electrophoresis on two identical gels. Subsequently, the proteins were transferred on PVDF membrane and incubated with [¹²⁵I]-C3b (A). Several distinct protein spots showing strongest binding of [¹²⁵I]-C3b were cut out from corresponding GelCode-stained gel (B) and subjected to mass spectrometry analysis (Table I). Two of the identified proteins, FACIN (acetylornithine aminotransferase) and GluD (glutamate dehydrogenase), were expressed recombinantly with His-Tags and purified by affinity chromatography to high purity as shown after separation of proteins by electrophoresis followed by Coomassie staining (C). (D) FACIN, GluD as well as positive (UspA2) and negative (α1-AT) controls were immobilized in microtiter plates and incubated with 3–50 μg/ml C3met. The binding was then detected with specific antibodies. (E) Surface plasmon resonance analysis of interaction between FACIN and C3b. C3b was immobilized on a CM5 sensor chip and FACIN at increasing concentrations ranging from 30 to 1000 nM was injected. Affinity constants were fitted using 1:1 Langmuir interaction model, shown are means of two independent measurements. The analysis of C3met binding to FACIN revealed a strong interaction with KD=8.9*10⁻⁸ M. (F) FACIN is located on the surface of F. alocis. Additionally, recombinant FACIN added to the bacteria showed a trend toward binding to the surface. FACIN was detected using specific antibodies using flow cytometry analysis. Means of three independent experiments are presented with bars indicating SD. Statistical significance of observed differences in binding (compared to negative control) was estimated using either a repeated measures (D) or a standard (E, F) two-way analysis of variance ANOVA (yielding p = 0.0153) and a Bonferroni post-test (to indicate exact differences at a given concentration); **** p<0.0001, ** p<0.01, * p<0.05, if nothing indicated – non-significant.

Fig. 5. F. alocis complement inhibitor FACIN destroys bactericidal activity of human serum

A) Classical pathway. NHS (0.15%) was supplemented with various concentrations of F. alocis proteins FACIN and GluD, BSA (negative control) or DAF-Fc (positive control) and preincubated for 15 min at 37°C after which sheep erythrocytes sensitized with antibodies and diluted in DGVB⁺⁺ were added. B) Alternative pathway. NHS (1.5%) was pre-incubated with increasing concentrations of proteins for 15 min at 37°C. Serum was then added to rabbit erythrocytes diluted in Mg⁺⁺-EGTA. For both A and B, after 1 h incubation, the degree of lysis was estimated by measurement of released hemoglobin (absorbance at 405 nm). Lysis obtained in the absence of any protein was set as 100%. C) E. coli DH5α were incubated with 0.75% NHS pretreated with increasing concentrations of proteins and the surviving bacteria were enumerated after overnight culture on LB agar plates. The survival was expressed as % of initial number bacteria in samples (% of inoculum). In A–C an average of three independent experiments is presented with bars indicating SD. Statistical significance of observed differences (compared to no protein added) was estimated using two-way repeated measures ANOVA (with p <0.0001 for all tests) and a Bonferroni post-test (to indicate exact differences at a given concentration); **** p<0.0001*** p<0.001 ** p<0.01, * p<0.05, if nothing indicated – non-significant.

Fig. 6. FACIN inhibits all pathways of complement

A–D) Proteins were preincubated with 1% NHS (A), 2% NHS (B, D) diluted in GVB⁺⁺ or 4% NHS in Mg-EGTA (C) and added to microtiter plates coated with IgGs (A, D), mannan (B) or zymosan (C). After 35 min (A–B) or 45 min (C–D) of incubation, the plates were washed and deposited C3b (A–C), and MAC (D) were detected with specific Abs. Absorbance obtained in the absence of any protein was set as 100%. An average of three independent experiments is presented with bars indicating SD. Statistical significance of observed differences compared to a condition in which no protein was added was estimated using two-way repeated measures analysis of variance ANOVA (with p < 0.0001 for all tests) and a Bonferroni post-test (to indicate exact differences at a given concentration); **** p<0.0001 *** p<0.001, ** p<0.01, * p<0.05, if nothing indicated – non-significant.

Fig. 7. FACIN inhibits alternative pathway convertase formation

A) NHS (5%) was pre-incubated with FACIN or DAF for 15 min on ice, and subsequently complement was activated in the samples by adding zymosan for 0, 10, 20 or 30 min. Reactions were stopped by adding reducing sample buffer and boiling for 5 min. Samples were then centrifuged and supernatant from the samples were separated by SDS-PAGE followed by blotting the proteins onto PVDF membrane. FB conversion to Ba and Bb was detected with specific pAbs. As a control, convertase was formed from purified components. B) F. alocis was incubated with increasing NHS concentrations in the presence of BSA or FACIN for 20 min at 37°C. Bacteria were then washed, suspended in reducing sample buffer and boiled. The amount of surface-attached FB/Bb was evaluated by western blotting with specific pAbs.

Fig. 8. FACIN inhibits deposition of C3b on F. alocis and inhibits phagocytosis

A) F. alocis was incubated for 45 min with 5% NHS or ΔNHS diluted in GVB⁺⁺. NHS was also supplemented with 3–100 μg/ml of FACIN. Deposited C3b was detected using specific antibodies and flow cytometry. Deposition/binding of C3b/C3 is shown as geometric mean fluorescence intensity (GMFI). B) Zymosan beads conjugated with fluorescein were opsonized with NHS in the absence or presence of FACIN, and subsequently added to differentiated HL-60 cells at 2:1 ratio for 1 h at 37°C. The uptake of fluorescent zymosan particles was evaluated by flow cytometry and the frequency of positive cells was calculated. The data were normalized to the signal obtained in NHS in each experiment. In A–B an average of three independent experiments is presented with bars indicating SD. Statistical significance of observed differences compared to NHS was estimated using one-way analysis of variance ANOVA (yielding p < 0.0001 in both (A) and (B)) and a Dunnett’s post-test; **** p<0.0001, ** p<0.01, * p<0.05.