Abstract
Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.
Introduction
Streptococcus pyogenes is a commonly encountered and clinically important pathogen (1). Every year, S. pyogenes infects ∼700 million people globally and causes life-threatening invasive infections in addition to mild superficial infections, such as impetigo and pharyngitis (1–4). S. pyogenes is one of the 10 most fatal human pathogens with ∼500,000 deaths annually (1). In most individuals, S. pyogenes affects the skin or oropharynx, but in some instances (∼650,000 cases worldwide annually), S. pyogenes invades deeper tissues, causing septicemia and/or necrotizing fasciitis. S. pyogenes binds specifically to human plasma proteins and thus evades human immune defenses in particular. Host proteins that bind to S. pyogenes include albumin, fibronectin, all four subclasses of IgG and the complement inhibitors C4b-binding protein (C4BP), and factor H (FH) (5–12). Other immune evasion mechanisms include sequestration of cathelicidin, enhanced survival in neutrophil extracellular traps, secretion of proteases and nucleases, and evasion of autophagy that promotes intracellular growth of S. pyogenes (13–17).
Complement plays an important role in combating S. pyogenes infections. Upon activation, the complement cascade generates inflammatory anaphylatoxins and deposits protein fragments onto foreign surfaces, which enable recognition of pathogens by professional phagocytes (18). Complement activation must be tightly regulated to prevent unwanted damage to host cells, which is achieved by surface-bound as well as soluble complement inhibitors such as C4BP and FH. However, several pathogens, including S. pyogenes, have evolved to bind complement inhibitors and evade complement activation to prevent their subsequent elimination (19, 20).
S. pyogenes surface-associated virulence factors include M proteins and M-like proteins such as protein H (21, 22). Although more than 220 variants of the M protein have been identified so far (23), bacteria of the M1 serotype are the most prevalent worldwide (24). Protein H, an IgG Fc−binding virulence factor, presents exclusively on M1-expressing S. pyogenes strains and forms complexes with IgG such that IgG cannot activate complement or facilitate opsonophagocytosis, thus rendering them immunologically effete (25, 26). In addition to its ability to bind to several serum proteins (6, 7, 21, 26), protein H can also form homodimers (27, 28). Competition between C4BP and human (hu)-IgG for binding to protein H has been suggested (6, 8). In this study, we characterized the interactions between hu- IgG, -C4BP, and -protein H and reported a novel virulence mechanism of S. pyogenes.
Materials and Methods
Bacteria and culture conditions
S. pyogenes AP1, AP4, AP8, AP15, AP18 (29), AP28, AP29, AP36, AP38, AP43, AP46, AP60, and AP74 (all from the World Health Organization Collaborating Centre for Reference and Research on Streptococci, Prague, Czech Republic) and AP1 isogenic mutants MC25 (M protein−) (30), BM27.6 (protein H−) (25), BM27.6 + pH (31), and BMJ71 (protein H−/M−) (32) were grown in Todd-Hewitt broth overnight at 37°C and 5% CO2. Cultures were then diluted to OD600 = 0.1 in fresh Todd-Hewitt broth and further incubated at 37°C in 5% CO2 and grown to OD600 of 0.3–0.4. Prior to use, bacteria were washed with PBS. Strains used are listed in Supplemental Table I.
Proteins and Abs
For flow cytometric analysis, the following Abs were used: mouse anti–hu-C4BP MK104 (33) coupled to biotin; mouse anti–hu-FH MRC OX24 (34) coupled to DyLight 647; goat anti–hu-f(ab)2 (Hycult); donkey f(ab)2 anti-rabbit IgG coupled to AF647 (Jackson ImmunoResearch); donkey f(ab)2 anti-goat IgG coupled to AF647 (Jackson ImmunoResearch); rabbit anti–mouse-C4BP (made in-house) coupled to DyLight 647; and mouse anti–mouse-FH (Hycult) biotin conjugated. Biotin-coupled Abs were stained with streptavidin-PE (eBioscience).
Fab and Fc fragments of hu-IgG were purchased from Calbiochem, and hu-IgG (IVIG; Kiovig) was purchased from Baxalta. Mice were administered pooled hu-Fc fragments (Athens Research), IVIG, or denosumab (Amgen) diluted in PBS. Rabbit IgG was purified from preimmune serum using protein A/G columns. Mouse IgG2a and IgG2b were purchased from ImmunoTools, and goat control IgG was purchased from R&D Biosystems. Goat, rhesus, and cynomolgus IgG were purchased from Nordic Diagnostica. hu-C4BP and FH were purified from human plasma, M18 was purified from culture supernatants using fibrinogen Sepharose, and Enn18 and protein H were expressed and purified from E. coli, all according to previously described protocols (8, 35). α1-Antitrypsin (α1AT) was used as a negative control for binding experiments. Plasma-purified C4BP preparations of 2 mg/ml contained between 2 and 10 μg/ml hu-IgG, as determined by a sandwich ELISA for hu-IgG.
Binding of [125I]protein H, [125I]C4BP, [125I]Enn18, and [125I]M18 to purified proteins
Purified proteins (C4BP, FH, α1AT, fibrinogen [American Diagnostica], human serum albumin [Sigma], or fibronectin [Haematologic Technologies]) were diluted to specified concentrations in PBS and immobilized onto microtiter plates (MaxiSorp BreakApart; Nunc) at 4°C overnight. The plates were washed three times with wash buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% Tween 20), and nonspecific binding sites were blocked with 3% fish gelatin (Norland Products) in wash buffer. 125I-labeled protein H, Enn18, or M18, respectively, were diluted in binding PBS (PBS supplemented with 0.1% Tween 20 and 0.1% BSA) and added in the presence of increasing amounts of IVIG. After incubation at 4°C overnight and subsequent washing, radioactivity in the wells was detected using a Wizard2 γ counter (PerkinElmer).
Binding of [125I]C4BP to S. pyogenes
125I-labeled C4BP, diluted in binding PBS, was added to bacteria either in the presence or absence of indicated amounts of IVIG. After 1 h incubation at 37°C (if not stated otherwise) in 5% CO2, bacteria were washed three times in 1× PBS, and radioactivity associated with bacteria was detected using a Wizard2 γ counter (PerkinElmer).
Electron microscopy
The presence and location of individual molecules or as molecular complexes on bacterial surfaces were analyzed by negative staining and transmission electron microscopy, as described previously (36). To visualize protein complexes, C4BP and protein H were coincubated in the presence or absence of 1 mg/ml IVIG for 1 h at 37°C. To detect C4BP binding to bacteria, C4BP and IVIG were conjugated with colloidal gold (Au). Bacteria were either stained with Ab-Au conjugates or mixed with protein-Au conjugates and incubated for 1 h at 37°C. Five milliliter aliquots were adsorbed onto carbon-coated grids for 1 min, washed with two drops of water, and stained with two drops of 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were examined using a Philips/FEI CM 100 electron microscope operated at an accelerating voltage of 80 kV; images were recorded with an Olympus Soft Imagining Solutions Veleta and side-mounted digital slow scan 2k × 2k CCD camera system using DigitalMicrograph software. The area of protein complexes was measured in Adobe Photoshop CS6. Proteins that were in closer contact than 30 nm or less were considered to interact or to be colocalized. Contrast, brightness, and pseudocolor enhancement were adjusted using Adobe Photoshop CS6.
Survival analysis
All animals were housed and bred under specific pathogen-free conditions in the animal facility at the University of Massachusetts Medical School, Worcester, MA. All experimental groups were sex- and age-matched (6–8-wk-old male and female BALB/c animals and hu-C4BP transgenic [tg] BALB/c).
One day prior to infection, animals were treated either with 1 mg hu-IgG–Fc, 2 mg IVIG or a monoclonal hu-IgG (denosumab), a hu-IgG2 monoclonal Ab that only recognizes human but not mouse RANKL) (37). As negative controls, either sterile PBS or 2 mg goat IgG was used. Animals were infected intravenously via lateral tail vein injection with 100 μl bacterial suspensions in PBS containing S. pyogenes AP1 at indicated concentrations (29). hu-IgG injections were repeated either every third day (1 mg hu-IgG–Fc per animal) or once on day 2 (0.5 mg IVIG, denosumab, or goat IgG). All animals were closely monitored for signs of disease for up to 8 d; gravely moribund mice were euthanized.
Serum preparation
Animals were anesthetized with Isoflurane, and blood was collected by cardiac heart puncture. Blood samples were kept on ice for 30 min and allowed to clot before centrifuging for 10 min at 1700 × g, 4°C. Serum was separated, aliquoted, and frozen immediately at −80°C until use.
Complement deposition and IgG-binding assays
Bacteria were incubated with increasing amounts (0.1–5%) of normal or hu-C4BP tg mouse serum or indicated IgG preparations for 1 h at 37°C in 5% CO2, if not stated explicitly otherwise. For testing the effect of temperature on C4BP binding, we added 150 μg/ml kanamycin, an inhibitor of protein biosynthesis, to all buffers to prevent alterations in the transcriptome due to temperature changes. Bacteria were washed thrice with PBS before and after each staining step. Bacteria were stained to detect surface-bound hu IgG, hu or mouse C4BP, or FH and then analyzed using a CytoFLEX (Beckman Coulter) or a CyFlow Space flow cytometer (Partec).
Polymorphonuclear cell–killing assay
Human polymorphonuclear cells (PMNs) were isolated on a Histopaque and a discontinuous Percoll gradient as described (38). In a 96-well plate, 1 × 105 PMNs per well were infected with S. pyogenes strain AP1 and AP18 at a multiplicity of infection of 0.1 in the presence of hu-C4BP tg mouse serum and hu-IgG. PMNs were incubated at 37°C, 5% CO2 for indicated times. Fifty microliters of the PMN bacteria mixture were diluted serially in PBS, plated onto blood agar, and incubated overnight at 37°C and 5% CO2 to enumerate surviving S. pyogenes.
Identification of protein H in whole genome sequences
The whole genome raw sequence data for 3615 S. pyogenes strains from BioProject PRJNA236767 was downloaded from the NCBI Short Read Archive, consisting of 3615 runs (accession number SRA036051; https://www.ncbi.nlm.nih.gov/sra?term=SRA036051), using the fastq-dump tool from the Short Read Archive Toolkit version 2.5.2 (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software). The raw data were mapped to a reference genome containing the protein H gene (NCTC 8198, accession number LN831034.1) using the high-sensitivity aligner SMALT version 0.7.5. First, the median coverage over the whole genome was determined using genomecov from bedtools version 2.23.0 (39). The coverage of the unique part of protein H (genomic position 1758555–1759380) was determined using SAMtools Mpileup version 0.1.19 (http://www.htslib.org), which recorded the number of bases covered and the average depth of coverage of the whole region. Because protein H amino acid sequences are believed to be hypervariable across strains, an additional search was performed to capture protein sequences that were sufficiently divergent from the reference genome that SMALT was not able to align to them. Using RAPsearch version 2.23 (40) (http://sourceforge.net/projects/smalt/), the data from all samples were mapped against protein sequences of the reference genome, outputting all hits. Every sequence read that had a better bit-score to protein H than to any other protein, and with at least 50% identity, was considered a potential hit.
Size exclusion chromatography
Size exclusion chromatography was performed using an ÄKTAexplorer System (GE Healthcare), employing a Superose 6 10/30 column using PBS as eluent, with 0.6 ml/min flow at ambient temperature. Proteins were incubated together in PBS for 30 min at room temperature before analysis. Nonmixed protein served as controls. Proteins were injected in 200 μl PBS, and the absorbance at 280 nm was recorded to identify the elution profile.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0b. To test for significance, we used one-way or two-way ANOVA analysis with Bonferroni’s posttest or Mantel Cox (log-rank; to analyze survival) tests as indicated. A p value < 0.05 was considered to be significant. Sample sizes in animal experiments were chosen to achieve statistical power while minimizing animal use.
Human wound sample
Necrotic tissue was collected from a subject with necrotizing fasciitis (in 2006) in whom S. pyogenes M1 was identified as the sole pathogen by the clinical microbiology department of Skåne University Hospital in Lund, Sweden. Although sequence verification of the organism was not performed at that time, the M protein and protein H were identified immunohistochemically.
Study approval
The use of animals in this study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health and the Swedish Animal Welfare Act SFS1988:534. All animal experiments were approved either by the Institutional Animal Care and Use Committee at the University of Massachusetts Medical School or by the Laboratory Animal Ethics Committee of Malmö/Lund, Sweden. The ethics committee in Lund approved the use of the human wound material and written informed consent was obtained from human subjects.
Results
hu-IgG augments protein H–C4BP binding
S. pyogenes M proteins are known to bind human serum proteins, especially complement inhibitors and Igs (41–49). In earlier publications, we showed that surface-bound C4BP enhanced virulence of S. pyogenes (8, 29). In this study, we sought initially to determine the importance of C4BP binding to S. pyogenes more generally in the presence of other human serum components, particularly hu-IgG. We tested 12 different S. pyogenes isolates that expressed different M proteins (Fig. 1A). Surprisingly, we observed that IgG binding increased C4BP binding in 5 of 12 (42%) M types. All strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. We chose to characterize the IgG–C4BP interaction on the M1 type strain, AP1 in particular, because this strain is well characterized and is virulent in hu-C4BP tg mice (6–8, 12, 21, 26–29, 35). AP1 binds C4BP and IgG via protein H, a member of the M protein family. We examined 3465 different M1 strains for protein H and found that 30% carried sph, the gene that encodes for protein H (Fig. 1B). The frequency of sph has been similar in its geospatial distribution (Fig. 1C; except Iceland) and across invasive and pharyngeal isolates (Fig. 1D, 1E) during the past 50 y, here represented by older preresurgence strains before 1980 (Fig. 1F) (50) and more contemporary postresurgence strains (Fig. 1G) (51, 52).
C4BP binding in S. pyogenes strains and presence of sph in M1. Twelve different S. pyogenes clinical isolates were tested for their C4BP binding in the presence and absence of hu-IgG (IVIG). Five strains showed significantly increased C4BP binding if coincubated with IgG (A). Full genome sequences from 3645 S. pyogenes M1 isolates were tested for the presence of sph. (B) 1055 out of 3645 isolates contain the gene for protein H, sph. (C) Geospatial analysis revealed that 28.91 ± 5.4% of all isolates from different countries have sph. Only Iceland with 72% appears as an outlier, possibly due to low sample numbers. (D–G) A similar frequency of sph was found in invasive strains [(D), 850 out of 2866 isolates] and in pharyngeal isolates [(E), 203 out of 597 isolates]. Interestingly, the distribution of 30% of sph+ strains was similar (31.98 and 30.37%, respectively) among SF370 [(F), 55 out of 172 isolates] and 5005-like M1 strains [(G), 1000 out of 3293 isolates], representing older preresurgence strains before 1980 and more contemporary postresurgence strains, respectively. Our data suggest that protein H has not emerged recently, but likely has persisted in about one-third of all M1 strains for at least the past 50 y. Statistical significance was calculated using a two-way ANOVA with Bonferroni's multiple comparison. Absence of asterisks indicates no significance. *p < 0.05, **p < 0.01, ****p < 0.0001. gMFI, geometric mean fluorescence intensity.
The binding site for C4BP on protein H resides in close proximity to the site where hu-IgG binds (Fig. 2A), suggesting competition between C4BP and IgG binding to protein H (8). Binding of increasing concentrations of 125I-labeled protein H to immobilized C4BP was measured in the presence or absence of 25 μg/ml hu-IgG (IVIG) at 37°C. IgG enhanced binding of protein H to C4BP at all concentrations of protein H tested. In the absence of IgG, a 100-fold increase in protein H concentration was required before any binding to C4BP was noted (Fig. 2B). Incubation at 4°C, which induces protein H dimerization (27), increased the amount of protein H bound to C4BP at identical protein H concentrations (Fig. 2C). IgG enhanced C4BP–protein H interactions, even at 37°C (Fig. 2B); therefore, we reasoned that lower temperatures and IgG each increased C4BP binding, possibly by di-/multimerization of protein H.
Binding of S. pyogenes protein H to hu-C4BP: effect of hu-IgG and temperature. (A) Schematic representation of protein H and binding sites in protein H for its known ligands (numbers indicate amino acid positions, references in superscript). (B) Immobilized C4BP was incubated with increasing amounts of [125I]protein H at 37°C in the presence or absence of 25 μg/ml IVIG. (C) Increasing amounts of [125I]protein H were added to immobilized C4BP at 4 or 37°C. (D) hu-IgG increases [125I]protein H binding to immobilized C4BP at 4, 30, and 37°C. [125I]protein H (250 kcpm) was incubated with increasing amounts of hu-IgG. (E) [125I]protein H (5–270 kcpm) was incubated with increasing amounts of hu-IgG and analyzed for binding to albumin, FH, fibronectin, or α1AT as a negative control. (F) [125I]Enn18 was incubated with increasing amounts of hu-IgG and tested for binding to different serum proteins in the presence of different amounts of hu-IgG. (G) Immobilized C4BP (1 μg/ml) was incubated with [125I]protein H (125 kcpm) in the presence of increasing amounts of whole hu-IgG, Fc, or Fab fragments. (H) Immobilized C4BP was incubated with [125I]protein H (125 kcpm) in the presence of increasing amounts of IgG from either human, rabbit, mouse, or goat. (I) S. pyogenes AP1 was incubated with 1 mg/ml IgG from the indicated species in 10% mouse serum containing hu-C4BP. Mean (± SD) from three independent determinations are shown in all experiments. Curve comparison for differences was performed using a two-way ANOVA: p < 0.0001 (B, F, and H), p = 0.0001 (D and E), and p = 0.002 (C). MFI = mean fluorescence intensity.*p < 0.05, **p < 0.01, ***p < 0.001 assessed by one-way ANOVA.
We next examined the effects of increasing concentrations of IgG on the binding of [125I]protein H to immobilized C4BP at different temperatures (Fig. 2D). IgG enhanced binding of protein H to C4BP at all three temperatures in a (IgG) dose-dependent manner.
Protein H–hu-IgG interaction selectively enhances C4BP binding to protein H
We asked if the protein H–hu-IgG interaction affected the binding of other protein H ligands (Fig. 2A). [125I]protein H was incubated with immobilized albumin, FH, fibronectin, or α1AT in the presence of IgG, except for albumin, which showed a decrease in protein H binding, and no increase in bound concentration to any of the protein H ligands was measured in the presence of increasing concentrations of IgG (Fig. 2E). Thus, hu-IgG specifically, increased the binding of protein H only to C4BP (Fig. 2D, 2E). It is worth noting that the y-axes in Fig 2D and 2E differ; the amount of protein H binding to C4BP at 4°C, in the presence of 10 μg/ml of IgG, was one to two orders of magnitude greater than binding to other protein H ligands.
We also examined the influence of IgG on the binding of C4BP, FH, albumin, and α1AT to Enn18, an M protein family member of M18 strains (49). [125I]Enn18 binding to C4BP did not increase because of IgG, whereas no binding to FH, albumin, or α1AT was observed (Fig. 2F). Taken together, IgG interacts with protein H to specifically increase the amount of C4BP that is bound but does not increase affinity for any of the other ligands tested.
To identify the IgG region containing the binding sites for protein H, we incubated [125I]protein H with immobilized C4BP in the presence of increasing amounts of intact IgG or corresponding concentrations of Fc or Fab fragments (Fig. 2G). Complex formation resided exclusively in hu-Fc–containing fragments. Non–hu-IgG did not increase the affinity of protein H for hu-C4BP (or to a low extent in the case of rabbit IgG; Fig. 2H). Similar to hu-IgG, nonhuman primate IgG increased the amount of C4BP that bound to AP1 (Fig. 2I).
hu-IgG forms complexes with protein H and C4BP
To verify the observed protein–protein interaction, we performed size exclusion chromatography of C4BP–protein H, C4BP–IgG, and protein H–IgG complexes. Compared to the individual proteins, C4BP and protein H formed a complex with a corresponding reduction in the free protein H peak, judged by the different elution volumes (Fig. 3A). In contrast, C4BP and IgG did not interact (Fig. 3B). Protein H–IgG bound each other, resulting in a new peak (Fig. 3C).
Complex formation of C4BP, protein H, and hu-IgG. (A) C4BP-protein H, (B)C4BP–IgG, and (C) protein H–hu-IgG as well as individual proteins were analyzed by size exclusion chromatography on a Superose 6 column. (D and E) Electron microscopy images of negative-stained protein complexes formed between hu-IgG, protein H, and C4BP. Proteins were artificially colored: C4BP, yellow; protein H, green; and IgG, red. Scale bars, 50 nm. Representative experiments of at least three consistent repetitions are shown.
Furthermore, we visualized complex formation between the three proteins using electron microscopy. Protein H was incubated with C4BP either in the absence (Fig. 3D) or presence (Fig. 3E) of IgG. In the absence of IgG, we found only ∼7% of C4BP molecules (yellow) complexed with protein H (green). In the presence of IgG (red), 83% of C4BP molecules were complexed with protein H.
S. pyogenes binds larger amounts of C4BP in the presence of IgG
We incubated AP1 and its isogenic mutant strains MC25, BM27.6, and BJM71 with 125I-C4BP in the presence or absence of IgG (Fig. 4A). AP1 and its isogenic mutant MC25 (both expressing protein H) bound C4BP, which increased significantly in the presence of IgG. Mutant strains BM27.6 and BMJ71, lacking protein H, did not bind C4BP independent of the presence of IgG. Complementing protein H in BM27.6 (BM27.6 + pH) restored increased C4BP binding due to IgG (Fig. 4B).
Hu-IgG increases C4BP-binding to bacteria. (A) S. pyogenes strains AP1 and isogenic mutants MC25 (protein H−), BM27.6 (protein M−), and BMJ71 (protein M−/H−) were incubated with [125I]C4BP (100 kcpm) in the presence or absence of 1 mg/ml hu-IgG. (B) AP1, BM27.6, and protein H–complemented mutant BM27.6 + pH were incubated in 10% mouse serum containing hu-C4BP in the presence or absence of 1 mg/ml hu-IgG. (C) AP1 was incubated with [125I]C4BP (100 kcpm) and increasing amounts of hu-IgG at 37°C. (D) AP1 was incubated within 10% mouse serum containing hu-C4BP in the presence or absence of 1 mg/ml hu-IgG at the indicated temperatures. (E) Electron microscopic confirmation of increased C4BP binding to AP1 in the presence of hu-IgG. (F) Hypothetical model of IgG-induced binding of C4BP to protein H. Mean (± SD) from three independent determinations are shown. More than 500 interactions from different areas of the microscopy grids were analyzed. Scale bars, 100 nm. **p < 0.01, ***p < 0.001, ****p < 0.0001 assessed by one-way (C) or two-way (A, B, and D) ANOVA.
We analyzed the influence of different IgG concentrations on C4BP binding to S. pyogenes AP1. C4BP binding to AP1 was maximal in the presence of 1 mg/ml of IgG (Fig. 4C). At 10 mg/ml IgG, C4BP levels decreased to levels similar to that seen in the absence of IgG.
C4BP binding to intact bacteria in the presence of IgG was not affected by temperature (Fig. 4D). In the absence of IgG, however, we found that increasing temperature significantly decreased C4BP binding capacity of AP1 from 40% at 30°C to <10% at 39°C, compared with binding of C4BP to bacteria in the presence of IgG across these temperatures.
Using gold-labeled C4BP (10 nm) and IgG (5 nm), we visualized the binding of C4BP to AP1 by electron microscopy (Fig. 4E). In the presence of IgG, we noted more C4BP (917 ± 84 gold particles/mm2) bound to the surface than without IgG (82 ± 8 gold particles/mm2). The majority of bound C4BP and IgG were in close proximity. No binding to bacteria was observed with control polyethylene glycol–coated gold particles of the same size (Supplemental Fig. 1A–C). We hypothesize that protein H di/multimerizes in the presence of hu-IgG, which permits greater binding to C4BP (Fig. 4F).
Hu-IgG increases the amount of C4BP that binds to bacterial surface proteins and prevents opsonophagocytic killing
We compared C4BP binding to S. pyogenes AP1 and AP18 in the presence of IgG. Only AP1, but not AP18, showed a significant increase in bound C4BP consistent with the observed effect of IgG to purified protein H and Enn18 (Fig. 5A).
Hu-IgG enhances C4BP binding to bacteria and promotes bacterial survival. To assess complement deposition, different S. pyogenes strains were incubated in hu-C4BP tg mouse serum and analyzed for C4BP binding (A) in the presence and absence of hu-IgG. Survival assay of S. pyogenes AP1 (B) and AP18 (C) coincubated with isolated PMNs and hu-C4BP tg mouse serum in the presence and absence of hu-IgG. Mean (± SD) from three independent determinations are shown. Statistical significance was calculated using a two-way ANOVA with Bonferroni’s multiple comparison. Absence of asterisks indicates no significance. **p < 0.01, ***p < 0.001. gMFI, geometric mean fluorescence intensity.
Next, we assessed the influence of IgG on the killing of S. pyogenes by PMNs. Addition of IgG to AP1 significantly increased survival at 60 and 90 min (Fig. 5B) but did not alter survival of AP18 (Fig. 5C).
Taken together, these data show that hu-IgG increases the amount of C4BP that binds to surface AP1, but not AP18, of S. pyogenes, which reduces opsonophagocytic killing.
Enhancement of C4BP binding to S. pyogenes by hu-IgG increases lethality in mice
AP1 does not bind mouse C4BP or FH (Supplemental Fig. 2A–B). In contrast, in hu-C4BPxFH tg mouse serum, binding of hu-C4BP was augmented ∼2-fold upon adding 1 mg/ml hu-IgG to the mouse serum (Supplemental Fig. 2C), whereas binding of hu-FH in tg mouse serum was not increased upon hu-IgG addition (Supplemental Fig. 2D). Interestingly, analysis of binding curves (Supplemental Fig. 2C) revealed that adding IgG does not increase the affinity (KD) of C4BP for AP1, compared with analysis when IgG is absent. However, maximal binding of C4BP to AP1 nearly doubled when IgG was present, indicating more C4BP binding sites on the bacteria in the presence of IgG.
To exclude the effect of Fab-directed opsonization in vivo, we used IgG-Fc fragments. Hu-C4BP tg mice were treated 1 d prior to infection (Day −1), either with 1 mg IgG-Fc or mock treated as a control. All infected animals treated with IgG Fc succumbed to infection by Day 4 (Fig. 6A). Mock-treated and infected animals survived significantly longer. Injection of 1 mg of Fc on Days −1, 2, and 5 yielded peak serum concentrations of up to 1.8 μM (Supplemental Fig. 3A). Fc increased C4BP binding to AP1, similarly to whole hu-IgG (Fig. 6B).
Hu-IgG increases lethality of S. pyogenes infection in hu-C4BP tg mice. (A) Hu-C4BP tg animals were injected i.p. with either 1 mg hu-IgG–Fc (n = 13) or mock treated (n = 12) 24 h prior to i.v. infection with 1.5 × 107 S. pyogenes AP1. (B) hu-IgG–Fc fragments exhibit similar effects as intact hu-IgG (IVIG) on C4BP binding to S. pyogenes. (C and E–H) BALB/c or hu-C4BP tg animals were injected i.p. with either 2 mg denosumab, IVIG, or mock treated 24 h prior to i.v. infection with S. pyogenes M18 or AP1 and monitored for 8 d. (C) Hu-C4BP animals (n = 10 per group) were treated with denosumab or mock treated, then infected with 1.25 × 107 S. pyogenes AP1. (D) C4BP binding to S. pyogenes in the presence of equivalent amounts of IVIG or denosumab. (E and F) Hu-C4BP tg mice (n = 10 per group) were given denosumab (E), IVIG (F), or mock treated, then infected with 4 × 107 S. pyogenes AP18. (G) Hu-C4BP tg mice (n = 10 per group) were given IVIG or mock treated, then infected with 2.5 × 107 S. pyogenes AP1. (H) BALB/c animals (IVIG n = 4 and PBS n = 5) were given IVIG or mock treated, then infected with 3 × 107 S. pyogenes AP1. Mean (± SD) from three independent experiments are shown (B and D). Control groups in (A) and (H) received PBS, and controls in (C) and (E)–(G) received goat IgG. Statistical significance was calculated using Mantel-Cox analysis (A, C, and E–H) and one-way ANOVA (B and D); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MFI, mean fluorescence intensity.
To determine if intact hu-IgG has similar effects on C4BP binding to S. pyogenes AP1, we used an unrelated hu-IgG2 mAb, denosumab. Neither denosumab nor goat IgG opsonized AP1 (Supplemental Fig. 3C). We pretreated hu-C4BP tg animals with 2 mg denosumab per animal on Day −1, to achieve serum hu-IgG levels of 6.6 μM (1 mg/ml). On Day 2, we reinjected 0.5 mg of denosumab, which led to peak serum levels of up to 9.0 μM (Supplemental Fig. 3B). Ninety percent of the animals treated with denosumab succumbed to infection, whereas all mock-treated animals survived (Fig. 6C). Similar results were achieved using a different AP1 inoculum (Supplemental Fig. 3D). Denosumab increased C4BP binding to AP1 in vitro (Fig. 6D), similar to hu-IgG, supporting the role of hu-IgG in enhancing AP1 infection.
To underline the importance of C4BP’s interaction with protein H in IgG-mediated virulence, we used S. pyogenes AP18, in which C4BP binding is not affected by hu-IgG (Fig. 5A). Administration of denosumab did not change mortality in AP18-infected mice compared with mock-treated animals (Fig. 6E). Similar to denosumab, AP18-infected hu-C4BP animals treated with hu-IgG (IVIG) showed no significant difference in survival compared with mock-treated animals (Fig. 6F). In contrast, IgG treatment of tg hu-C4BP mice prior to infection with AP1 significantly decreased their survival (Fig. 6G), similar to IgG-Fc (Fig. 6A) and denosumab (Fig. 6C).
Hu-IgG treatment of BALB/c WT mice did not affect survival compared with mock treatment during infection with AP1 (Fig. 6H, Supplemental Fig. 3E). Similarly, denosumab did not increase mortality of AP1-infected animals (Supplemental Fig. 3F) because S. pyogenes cannot recruit and use mouse C4BP. Goat IgG did not influence C4BP binding to bacteria (Fig. 1I) and thus did not alter the course of infection in mice compared with PBS treatment (Supplemental Fig. 3G).
Taken together, these data show a detrimental effect of hu-IgG on S. pyogenes infection in tg hu-C4BP–expressing mice.
IgG increases C4BP binding to S. pyogenes M1 in humans
We sought to verify the interaction of C4BP, IgG, and protein H in humans by analyzing tissue samples from a subject with necrotizing fasciitis caused solely by S. pyogenes that expressed M1 protein. Scanning and transmission electron micrographs from biopsy samples confirmed the presence of bacteria, indicated by white (Fig. 7A) and black (Fig. 7B) arrows. We stained these samples for C4BP, protein H, and IgG or M protein (Fig. 7C–G, Supplemental Fig. 1D–G). Because protein H expression is regulated during the course of infection, we found examples of S. pyogenes M1 with or without protein H in the same sample. Protein H–positive M1 bacteria bound >3.5 times more anti-C4BP–labeled gold particles than protein H–negative bacteria (273 ± 38 C4BP particles/μm2 versus 73 ± 21 C4BP particles/μm2; Fig. 7E). Colocalization analysis revealed that C4BP colocalized 79 ± 7% of the time with protein H and IgG (Fig. 7F, 7G), whereas it localized with protein H alone in only 32 ± 5% of instances in the absence of IgG (Fig. 7H). These data provide evidence that IgG enhances C4BP binding to S. pyogenes during human infection.
Colocalization of protein H, IgG, and C4BP in tissue samples from a patient with necrotizing fasciitis. (A) Scanning electron microscopy of surgically excised necrotic tissue from a subject with necrotizing fasciitis of the left shoulder. White arrows indicate streptococci in the tissue. (B) Transmission electron micrograph from the same sample shows bacteria (black arrows), which are subsequently stained with different Abs. (C, D, F, and G) Bacteria are stained either with Abs coupled to gold particles (size in brackets) that react with C4BP (15 nm), (C and D) M protein (10 nm) or (F and G) IgG (10 nm), and protein H (5 nm). (C) S. pyogenes M1 that express protein H showed more C4BP (273 ± 38 gold particles/μm2) compared with protein H–negative S. pyogenes M1 bacteria [(D), 73 ± 21 gold particles/μm2], quantified in (E). IgG colocalizes with C4BP in protein H–positive bacteria (F) but to a lesser extent on protein H–negative bacteria (G). (H) C4BP–protein H colocalization is increased in the presence of IgG (79 ± 7%) compared with the absence of IgG (32 ± 5%). Scale bars, 5 μm (A), 2 μm (B), or 100 nm (C–F). Mean (± SD) of more than 50 bacteria are shown (E and H).
Discussion
We have identified a novel virulence mechanism of S. pyogenes, namely that hu-IgG increases the amount of C4BP that binds to S. pyogenes protein H. Binding of IgG leads to dimerization of protein H on the bacterial surface (27). Clustered protein H is then able to bind larger amounts of C4BP than monomeric protein H. Thereafter, bound C4BP limits complement activation and reduces opsonization and bacterial elimination by phagocytes. IgG not only increases the amount of C4BP that binds to purified protein H in vitro, but it also increases binding of C4BP to bacteria in vivo, thereby reducing complement activation and opsonophagocytosis. We found that hu-IgG enhances S. pyogenes infection in mice. Consistent with our findings, we also colocalized IgG, C4BP, and protein H in human tissues from necrotizing fasciitis caused by S. pyogenes. This virulence mechanism may be particularly important in niches such as mucosal surfaces and interstitial fluids where the availability of C4BP is diminished. Furthermore, we have evidence that this mechanism is not limited to M1 strains that express protein H but also occurs in other M type strains, thereby extending and strengthening the significance of this observation. All S. pyogenes strains that we tested that bound C4BP in the absence of IgG showed significant increased C4BP binding in the presence of IgG.
Protein H belongs to the family of M proteins and is expressed exclusively in M1 strains of S. pyogenes, the most frequently expressed M protein serotype in the Western world and the main cause of invasive and often lethal streptococcal infections (24, 53) (including 1, 14, 24, 54–59). Protein H is expressed in 28% (18 out of 64) of M1 serotype that cause necrotizing fasciitis, according to an analysis (60) of isolates from a CDC surveillance study (53). We confirmed that 30% (1055/3465) of S. pyogenes M1 strains had the sph gene encoding protein H or a homolog (51).
Because low temperatures induce dimerization of protein H (27, 61) and because IgG also exhibited a similar effect on protein H–C4BP binding, we speculate that IgG also di-/polymerizes protein H. This synergistic effect was supported by electron microscopy showing that IgG increased the prevalence of C4BP–protein H complex.
Analysis of monomeric recombinant protein H revealed no measurable affinity for their putative ligands (27). Dimerization of protein H, induced by lower temperatures or by binding of hu-IgG Fc, permitted C4BP binding. However, on the bacterial surface, we found no effect of IgG on affinity for C4BP binding to protein H at any IgG concentration. Of note, maximal binding increased ∼2-fold at higher temperatures (e.g., 37°C) in the presence of IgG. As a consequence, protein H dimerized, creating more binding sites for C4BP. We propose that protein H–protein H dimers are stabilized under these conditions, thus allowing for greater binding of C4BP to protein H.
We demonstrated that the Fc region in hu-IgG increased C4BP–protein H interaction and that rabbit IgG also bound to protein H. This finding is consistent with previous observations that protein H binds to rabbit, baboon, and guinea pig IgG but not to rat, mouse, bovine, or equine IgG (21, 35). Orientation of IgG on the surface of S. pyogenes depends on IgG concentrations. Extravascular fluids for example have lower levels of C4BP and less IgG compared with serum (62). The concentration of C4BP bound to S. pyogenes was maximal at 1 mg/ml IgG (∼1/5th–1/10th of serum concentration in healthy adults; see Ref. 63), consistent with our assertion that S. pyogenes has adapted to bind C4BP in environments with low levels of both C4BP and hu-IgG, such as extravascular compartments. In IgG-poor environments, S. pyogenes binds IgG via the Fc region predominantly; in undiluted serum, the interaction is mediated mainly by specific IgG Abs binding to their antigenic targets on the streptococcal surface via Fab2 (41). S. pyogenes counteracts Ig-mediated opsonization by secreting enzymes that cleave and inactivate surface-bound IgG (2, 64, 65). It is believed that the binding of IgG Abs via effector Fc fragments to microbes renders them immunologically effete (66), e.g., by preventing Fc-receptors from recognizing IgG-opsonized bacteria (48). In this study, we provide evidence that IgG binding is indeed an immune evasion mechanism. C4BP and IgG share a common binding site at the C terminus of domain A and the N terminus of domain B (6, 8), and this could explain why we were unable to identify any strains among the 12 tested that bound to only one of these proteins.
C4BP bound to S. pyogenes increases adherence and invasion of endothelial cells (8). Thus, enhancement of C4BP binding to protein H in the presence of hu-IgG may facilitate bacterial invasion. The virulence mechanism described in this article explains how IgG enables S. pyogenes to bind C4BP at temperatures encountered in vivo; protein H alone does not recruit C4BP at these temperatures. However, SpeB, a cysteine protease secreted by S. pyogenes, cleaves protein H to release a 36 kDa fragment, which contains the IgG binding region (67). Recruitment of C4BP to the released protein H–IgG complex could further enlarge these immune complexes rapidly and contribute to microthrombus formation, glomerulonephritis, and acute renal failure, all of which are complications of invasive streptococcal infections (68, 69).
These data highlight a novel mechanism that certain strains of S. pyogenes may use to flourish in their natural ecological niches, the throat and skin, where lower levels of IgG and complement proteins exist. The results also emphasize the significance of complement inhibition in the pathogenicity of S. pyogenes infection, a phenomenon originally described for FH (5). Targeting interactions between bacteria and host complement inhibitors may offer new opportunities to treat invasive S. pyogenes infection.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank Oonagh Shannon (Lund University) and Nancy Nowak and Bo Zhang (both of University of Massachusetts Medical School) for expert help with breeding and caring for mice, Maria Baumgarten for her skillful technical assistance, and the Core Facility for Integrated Microscopy at the Panum Institute, Copenhagen University for help with electron microscopy.
Footnotes
This work was supported by Swedish Research Council Projects 2016-01142 and K2014-58X-07480-29-5, the Swedish Government Funds for Clinical Research, the Torsten Söderberg Foundation, the Crafoord Foundation, the Knut and Alice Wallenberg Foundation, the Lars Hierta Memorial Foundation, the Österlund Foundation, the Gustav V 80-Years Anniversary Foundation, the Gyllenstierna Krapperups Foundation, and National Institutes of Health Grants R01AI114790 (to P.A.R. and S.R.) and R21 AI111728 (to J.S. and S.R.). M. Magda was supported by the Department of Biotechnology at the University of Rzeszow, Poland. A.W. received funding from École Normale Supérieure, Paris, France.
The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- α1AT
- α1-antitrypsin
- C4BP
- C4b-binding protein
- FH
- factor H
- hu
- human
- PMN
- polymorphonuclear cell
- tg
- transgenic.
- Received January 23, 2018.
- Accepted March 12, 2018.
- Copyright © 2018 by The American Association of Immunologists, Inc.
References
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