Prominent Binding of Human and Equine Fibrinogen to Streptococcus equi subsp. zooepidemicus Is Mediated by Specific SzM Types and Is a Distinct Phenotype of Zoonotic Isolates

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins.

ABSTRACT

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.

INTRODUCTION

Streptococcus equi is a Gram-positive beta-hemolytic coccus belonging to Lancefield group C. The species is divided into S. equi subsp. equi and S. equi subsp. zooepidemicus. Whereas S. equi subsp. equi is essentially confined to equids (1), causing strangles, S. equi subsp. zooepidemicus is found in a wide range of animals and in humans. As an opportunistic pathogen of horses, S. equi subsp. zooepidemicus not only colonizes mucosal surfaces but also causes a wide variety of diseases, such as pneumonia, arthritis, abortion, and wound infections. It is the most important bacterial pathogen associated with pneumonia in adult horses (2). In 2010, a specific clone of S. equi subsp. zooepidemicus (sequence type [ST] 209) spread through the Icelandic horse population, leading to an unprecedented epidemic of respiratory disease affecting almost the entire horse population of 77,000 animals (3). In humans, S. equi subsp. zooepidemicus may cause bacteremia, meningitis, arthritis, or endocarditis (4–10). Many of the reported cases have been related to the consumption of unpasteurized milk products or to contact with horses (9, 11–13). S. equi subsp. zooepidemicus might cause clinical or subclinical mastitis in goats, sheep, and cattle (13–15). As subclinical mastitis might not be noticed, consumption of unpasteurized milk or respective cheese is a risk factor for this zoonosis (11, 13). In an example of a horse-related zoonosis, Pelkonen et al. (9) reported three unrelated cases of severe diseases in humans caused by S. equi subsp. zooepidemicus ST-10 and ST-209. All three patients had close contact with horses, and an S. equi subsp. zooepidemicus ST-10 strain was also isolated from a healthy horse in the stable of a patient who carried S. equi subsp. zooepidemicus ST-10.

S. equi subsp. equi and S. equi subsp. zooepidemicus share approximately 80% genome sequence identity with the human pathogen Streptococcus pyogenes (3, 16, 17). Fibronectin-, IgG-, and collagen-binding proteins as well as the hyaluronic acid capsule, extracellular nucleases, streptolysins, and superantigens are similar in these species. Both species harbor at least one dimeric coiled-coil surface anchored protein, called M or M-like protein. emm typing of S. pyogenes (the emm gene encodes the M protein) is commonly used to differentiate isolates (www.cdc.gov/streplab/index.html).

S. equi subsp. zooepidemicus harbors the M-like proteins SzM and SzP. SzM shows high heterogeneity (8). Recombinant SzM of strain NC78, which caused an epizootic of equine respiratory disease, binds equine fibrinogen and plasminogen (8), but loss of function experiments of S. equi subsp. zooepidemicus were not conducted and it is not known if binding of fibrinogen is a general characteristic of various SzM proteins. We asked if expression of SzM in S. equi subsp. zooepidemicus is necessary for recruiting host proteins to the bacterial surface and if differences in binding of equine and human fibrinogens among S. equi subsp. zooepidemicus isolates might be related to specific clusters of SzM and an increased zoonotic potential.

RESULTS

The zoonotic Streptococcus equi subsp. zooepidemicus isolate C33 binds human and equine fibrinogen.

Invasive streptococci such as S. equi subsp. zooepidemicus recruit host proteins to their surface. We hypothesized that S. equi subsp. zooepidemicus strains causing diseases in humans and horses should bind similar profiles of host proteins in both species. Other strains might have undergone evolutionary adaptation to horses as their main host. This should be associated with distinct phenotypes in the interaction with proteins of equine and human origins. To follow up this working hypothesis, we comparatively investigated the binding of equine and human plasma proteins to the surfaces of the S. equi subsp. zooepidemicus isolates C2 and C33 of equine and human origins, respectively (Fig. 1). In reducing SDS-PAGE, both isolates showed overall similar band patterns of proteins in the acidic eluates of the bacterial surfaces after incubation in plasma (Fig. 1A). These band patterns included a high-molecular-weight protein of >250 kDa, identified as fibronectin by Western blotting (Fig. 1D), several bands between 50 and 80 kDa, and a band at <25 kDa. The band patterns of proteins obtained from the bacterial surfaces were very similar for the equine and human isolates after incubation in equine plasma. However, after incubation in human plasma, proteins of around 60 kDa and 100 kDa were found only in the eluate of the human S. equi subsp. zooepidemicus isolate C33. Western blot analysis of the acidic eluates indicated that fibrinogen and IgG are among the host proteins binding to the bacterial surfaces of the two investigated S. equi subsp. zooepidemicus isolates C2 and C33 (Fig. 1B and C). The human S. equi subsp. zooepidemicus isolate C33 displayed a very prominent fibrinogen band around 70 kDa after incubation in human plasma (Fig. 1C). This band corresponds to the α-chain of human fibrinogen, which has a predicted molecular weight of between 63 kDa and 66.5 kDa. In contrast, only a small quantity of human fibrinogen was bound by the equine S. equi subsp. zooepidemicus isolate C2. Western blot analysis also showed that both strains bound equine fibrinogen, indicated by the detection of the α-chain around 75 kDa.

FIG 1.

The M-like protein SzM is necessary for fibrinogen binding of S. equi subsp. zooepidemicus isolates C2 and C33. The equine isolate C2 and its in-frame szm deletion mutant C2ΔSzM as well as the human isolate C33 and its in-frame szm deletion mutant C33ΔSzM were analyzed by plasma absorption assays. (A) Acidic eluates of isolates incubated with equine and human plasma were analyzed by reducing SDS-PAGE. Western blot analysis was conducted using anti-IgG (B), anti-human fibrinogen (specific for the α-chain) (C), or anti-fibronectin (D) antibodies. Molecular weights of the SDS-PAGE ruler are indicated at the left. Direct binding of fibrinogen of the S. equi subsp. zooepidemicus wt strains C2 and C33 as well as of their isogenic szm deletion mutants C2ΔSzM and C33ΔSzM was analyzed by flow cytometry using equine (E) and human (F) FITC-labeled fibrinogens. The amount of bound fibrinogen is shown as the geomean of fluorescence intensity (n = 3). eFg, equine fibrinogen; hFg, human fibrinogen.

The M-like protein SzM is crucial for fibrinogen binding of S. equi subsp. zooepidemicus isolates C2 and C33.

As a recombinant SzM protein was shown to interact with host proteins (18), we constructed isogenic in-frame deletion mutants of the szm genes in S. equi subsp. zooepidemicus isolates C2 and C33, designated C2ΔSzM and C33ΔSzM, respectively, and conducted loss-of-function experiments. Plasma absorption assays revealed that the band pattern of proteins in the acidic eluates was substantially reduced for the high-molecular-weight protein fibronectin (>250 kDa) (Fig. 1A and D) and a band of around 50 kDa in C2ΔSzM after incubation in equine plasma. This 50-kDa protein was identified as the heavy chain of IgG by Western blotting (Fig. 1B). In human plasma, the band pattern of proteins of C2ΔSzM was reduced for fibronectin (Fig. 1A and D). Similarly, the human plasma protein absorption assay with the mutant C33ΔSzM displayed, with the exception of very weak bands, only the high-molecular-weight protein fibronectin of >250 kDa (Fig. 1A and D). Thus, Western blot analysis demonstrated that SzM expression is necessary for binding of equine and human fibrinogens to the bacterial surface, at least in the observed amounts. SzM expression led to host-specific profiles of surface-bound plasma proteins distinct between the equine and the human isolates C2 and C33.

Furthermore, binding of equine and human fibrinogens to the bacterial surface was quantified by flow cytometry analysis using fluorescein isothiocyanate (FITC)-labeled fibrinogen. Equine fibrinogen was detected in comparable large amounts on the surfaces of S. equi subsp. zooepidemicus isolates C2 and C33, while the binding of equine fibrinogen was significantly attenuated in mutants C2ΔSzM and C33ΔSzM (Fig. 1E). The human S. equi subsp. zooepidemicus isolate C33 bound large amounts of human fibrinogen, which were significantly higher than for the equine isolate C2. The binding of human fibrinogen was significantly attenuated in both mutants C2ΔSzM and C33ΔSzM (Fig. 1F).

Based on these initial results, we hypothesized that SzM-mediated binding of human fibrinogen in large amounts to the bacterial surface is a hallmark of zoonotic S. equi subsp. zooepidemicus isolates.

Recombinant SzM of S. equi subsp. zooepidemicus isolate C33 binds Fc fragments of human IgG as well as equine and human fibrinogens.

SzM proteins of the human isolate C33 and the equine isolate C2 were expressed recombinantly as His-tagged proteins, designated rSzM_C33 and rSzM_C2, respectively, to investigate binding of host proteins to purified rSzM. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) revealed that rSzM_C33 and rSzM_C2 bind the Fc fragments of human IgG (Fig. 2). Human IgG-Fc fragments conjugated to horseradish peroxidase (HRP) showed direct binding to rSzM_C33 and rSzM_C2 but not to the control protein muramidase-released protein (rMRP) (Fig. 2A), which was detectable in an anti-His tag Western blot (Fig. 2B). Furthermore, rSzM_C33 and rSzM_C2 coated on ELISA plates showed concentration-dependent binding of the soluble ligand human IgG-Fc conjugated to HRP, while rMRP showed no interaction (Fig. 2C). Thus, antigen-independent binding of IgG prompted us to detect rSzM in binding of further host proteins with human IgG-Fc HRP.

FIG 2.

Recombinant proteins rSzM_C33 and rSzM_C2 bind Fc fragment of human IgG. (A) Western blot of immobilized rSzM_C33, rSzM_C2, and muramidase-released protein (rMRP) incubated with human IgG-Fc conjugated with HRP. (B) Anti-His tag Western blot of samples used in panel A. (C) ELISA plates were coated with doubling dilution series of rSzM_C2, rSzM_C33, or rMRP and probed with human IgG-Fc conjugated with HRP. IgG-Fc binding to recombinant proteins was detected after ABTS-H₂O₂ development via OD₄₀₅ measurements. The results shown are from three independent experiments. The Streptococcus suis protein rMRP was included as a negative control.

ELISAs with soluble rSzM_C33 (detected with human IgG-Fc HRP) as a ligand demonstrated binding of rSzM_C33 to immobilized equine as well as human fibrinogen in a dose-dependent manner (Fig. 3B and E). In contrast, the soluble ligand rSzM_C2 showed binding only to equine fibrinogen in a dose-dependent manner. rSzM_C2 did not show binding to human fibrinogen comparable to that of rSzM_C33 (Fig. 3A and D).

FIG 3.

rSzM of the human S. equi subsp. zooepidemicus isolate C33 binds equine and human fibrinogens, whereas rSzM of the equine S. equi subsp. zooepidemicus isolate C2 binds efficiently only equine fibrinogen. ELISA plates were coated with doubling dilution series of equine (A to C) or human (D to F) fibrinogens and probed with different amounts of rSzM_C2 (A and D) or rSzM_C33 (B and E). Binding of the soluble ligands to fibrinogen was detected by human IgG-Fc conjugated to HRP, ABTS-H₂O₂ development, and OD₄₀₅ measurement. For panels C and F, fibrinogen-coated ELISA plates were probed with 100 μl 0.5 μg/ml biotinylated rSzM_C2 and rSzM_C33 (rSzM_C2-biotin and rSzM_C33-biotin). Binding of the soluble ligands to fibrinogen was detected by streptavidin-HRP incubation and ABTS-H₂O₂ development and measurement of OD₄₀₅. eFg, equine fibrinogen; hFg, human fibrinogen. The results shown are from three independent experiments.

Binding of these recombinant proteins to fibrinogen was also investigated using biotinylated rSzM (designated rSzM_C2-biotin or rSzM_C33-biotin). Briefly, ELISA plates with immobilized equine or human fibrinogen were probed with rSzM_C2-biotin or rSzM_C33-biotin. Binding was detected with streptavidin-HRP. Again, whereas rSzM_C33-biotin bound equine as well as human fibrinogen in a dose-dependent manner, rSzM_C2-biotin bound only equine fibrinogen in a dose-dependent manner and did not show comparable binding of human fibrinogen (Fig. 3C and F). Thus, differences in fibrinogen binding between the two rSzM variants were confirmed through this independent approach.

The M-like protein SzM is necessary for survival in equine and human blood.

The ability to survive in blood is an important feature for any pathogen causing bacteremia and systemic diseases after dissemination in the blood. We hypothesized that loss of the SzM protein, associated with an attenuation in fibrinogen binding, would lead to increased killing of S. equi subsp. zooepidemicus in equine and human blood. Therefore, equine and human blood samples were infected with S. equi subsp. zooepidemicus wild-type strains and isogenic szm mutants ex vivo, and bacterial survival factors (SF), calculated by dividing the number of CFU after a 120-min incubation period at 37°C by the number of CFU at t = 0 min, were determined.

Wild-type C2, originally isolated from a horse, proliferated in the blood from every horse sampled (n = 8), resulting in a mean SF of 11.26 (standard deviation [SD], 2.56) (Fig. 4A; see also Fig. S3 in the supplemental material). In contrast, the isogenic mutant C2ΔSzM was very efficiently killed under the same conditions as indicated by an SF of 0.00625 (SD, 0.01). The specific bacterial load in equine blood was significantly lower in C2ΔSzM-infected than in C2 wild-type (wt)-infected equine blood after 120 min (Fig. S3). A significant difference in CFU in equine blood after a 120-min incubation was also observed in the respective comparison of C33 wt and its isogenic mutant C33ΔSzM (Fig. S3). Bacterial survival factors of C33 and its isogenic mutant C33ΔSzM in equine blood of 2.01 (SD, 2.58) and 0.1825 (SD, 0.23), respectively, were substantially but not significantly different (P = 0.0614) (Fig. 4A). However, in contrast to that with C2, the human C33 strain was reduced in number in the blood samples from 4 of 8 horses (Fig. S3).

FIG 4.

The M protein SzM of S. equi subsp. zooepidemicus is crucial for survival in equine and human blood. Blood from eight horses (A) or blood from six human donors (B) was ex vivo infected with S. equi subsp. zooepidemicus C2 wt strain, its isogenic mutant C2ΔSzM, wt S. equi subsp. zooepidemicus C33, or its isogenic mutant C33ΔSzM for 120 min. The indicated survival factors were calculated by dividing the number of CFU after a 120-min incubation period at 37°C by the number of CFU at t = 0 min. Strains C2 and C33 were originally isolated from a horse and a human, respectively. (C) Blood from 4 horses, reconstituted with plasma or serum, was ex vivo infected with S. equi subsp. zooepidemicus C2 wt or its isogenic mutant C2ΔSzM for 120 min. Briefly, blood cells were extensively washed with PBS to remove fibrinogen and finally resuspended in plasma (as a fibrinogen source) and serum (essentially no fibrinogen). CFU of C2 wt and its isogenic mutant C2ΔSzM were determined at time point zero and after 120 min of incubation. Resulting survival factors with SD are indicated. SEZ, S. equi subsp. zooepidemicus.

A whole-blood bactericidal assay with fresh blood from human volunteers (n = 6) revealed mean survival factors of S. equi subsp. zooepidemicus strains C2 and C33 of 4.17 (SD, 3.69) and 2.67 (SD, 1.53), respectively. The equine S. equi subsp. zooepidemicus strain C2 proliferated in the blood samples from 4 human volunteers under the chosen experimental conditions (Fig. S3), but the difference to strain C33 was not as pronounced as in equine blood (compare Fig. 4A and B). The survival of isogenic mutants C2ΔSzM and C33ΔSzM was attenuated in human blood as indicated by SFs of 0.35 (SD, 0.17) and 0.67 (SD, 0.69), respectively, which were both significantly lower than for the wt strains.

Additionally, the survival of S. equi subsp. zooepidemicus isolate C2 and its isogenic mutant C2ΔSzM was analyzed in a reconstituted equine blood bactericidal assay to determine the role of bacterial fibrinogen binding for survival. Briefly, washed blood cells were reconstituted with plasma and serum and infected ex vivo with S. equi subsp. zooepidemicus. In blood reconstituted with plasma, C2 wt reached significantly higher bacterial loads than its isogenic mutant C2ΔSzM after 120 min (Fig. 4C). No significant differences in the bacterial loads after 120 min were observed between C2 and its isogenic mutant C2ΔSzM when infecting equine blood reconstituted with serum, where essentially no fibrinogen is present. Thus, fibrinogen, supplied by plasma, leads to significant differences in the bacterial loads of C2 wt and the isogenic mutant C2ΔSzM, in which fibrinogen binding was found to be attenuated.

szm analysis of S. equi isolates.

Variability of M-like proteins is known to contribute to the diversity of S. equi subsp. zooepidemicus (8, 18). We asked if isolates with a zoonotic background might be associated with specific clusters defined by SzM sequences. Thus, the 5′ region of szm encoding the 216 N-terminal amino acids of the mature protein was used for phylogenetic analysis (Fig. 5). Meehan et al. showed that this region is important for fibrinogen binding in SeM of S. equi subsp. equi, since at least 188 amino acids of the N-terminal part of the mature SeM were crucial for binding of fibrinogen (19). At the least, the N-terminal part of rSzM_C33, comprising the N-terminal 216 amino acids of the mature protein, binds human IgG-Fc as well as equine and human fibrinogens (see Fig. S2).

FIG 5.

Phylogenetic analysis of amino acid sequences of the M-like proteins SzM. Two hundred sixteen amino acids of the N-terminal parts of the mature SzM proteins were used for phylogenetic analysis. Identified clusters are indicated. In addition to SzM sequences of this study, sequences of GenBank entries for Streptococcus equi (SzM_MGCS10565, ACG63105.1; SeM_4047, CAW95289.1; SzM_infant, AKL88101.1; SzM_H70, CAW97903.1; SzM_W60, AGE12544.1) and Streptococcus pyogenes (M1-protein, AAZ52337.1; Spa36, ACD81471.1) were included. •, equine isolates from Germany; ○, human isolates; S, Sweden; G, Germany.

In detail, PCR amplification products of szm genes with lengths ranging between 1,000 bp and 2,500 bp were generated from 35 S. equi subsp. zooepidemicus isolates with primer pair SeM/SzM_fwd and SeM/SzM_rev, indicating a high variability. This primer pair was also suitable to amplify the sem gene of S. equi subsp. equi. However, it was not possible to obtain amplification products with the above-described primer pair for the remaining 7 S. equi subsp. zooepidemicus isolates. Thus, primer pair SzM_down_fwd and SzM_up_rev was designed, leading to successful amplification and sequencing of the szm region of the remaining isolates.

One of the isolates (C68) showed a frameshift mutation in its szm gene, leading to a stop codon within the first 130 amino acids (aa) of the mature protein. Thus, it was excluded from phylogenetic analysis. Although the N-terminal part of SzM showed a high degree of diversity with 20 unique sequences of 39 analyzed isolates, 14 of the 25 (56%) human isolates grouped in three clusters, designated clusters I to III. Individual alignment analysis of SzM sequences of cluster I and cluster III revealed 70% amino acid similarity. SzM sequences of cluster II show 24% and 25% amino acid similarity to SzM sequences of cluster I and cluster III, respectively.

In contrast to the high diversity of SzM sequences of S. equi subsp. zooepidemicus, the M protein SeM of S. equi subsp. equi is conserved (cluster IV). Amino acid sequence similarity of SeM_4047 with SzM ranged between 26% (SzM_C12) and 50% (SzM_C30).

Binding of plasma proteins by various S. equi strains.

Forty-one S. equi subsp. zooepidemicus isolates of human (n = 24) and equine (n = 16) origin were investigated for plasma protein binding using equine and human plasma to test the hypothesis that the zoonotic background is associated with distinct phenotypes in the interaction with human host proteins. In general, equine isolates bound more equine plasma proteins than human plasma proteins (Fig. 6A and B). This was not the case for human S. equi subsp. zooepidemicus isolates. The patterns of proteins recruited to the bacterial surface did not differ in the human S. equi subsp. zooepidemicus isolates between equine and human plasma. Western blot analysis confirmed the presence of fibrinogen and fibronectin among the surface-bound proteins (Fig. 6C to F; Table 1).

FIG 6.

Representative SDS-PAGE gels and Western blots of plasma absorption assays of S. equi subsp. zooepidemicus. Acidic eluates of equine and human isolates incubated with equine (A) and human (B) plasma were analyzed by SDS-PAGE. Furthermore, samples were analyzed by anti-fibrinogen Western blot (C and D) and anti-fibronectin Western blot (E and F). Molecular weights of the SDS-PAGE rulers are indicated at the left. Faint Western blot signals as in panel D isolates C2 and C5 were interpreted as negative binding.

TABLE 1.

Binding of S. equi subsp. zooepidemicus isolates to fibrinogen and fibronectin in plasma absorption assays as detected by Western blotting

Isolates	No. binding/total no. (%)a
	Equine Fg	Human Fg	Equine Fn	Human Fn
S. equi subsp. zooepidemicus (human origin)b	19/24 (79.2)	20/24 (83.3)	24/24 (100)	24/24 (100)
S. equi subsp. zooepidemicus (equine origin)	8/17 (47.1)	6/17 (35.3)	17/17 (100)	17/17 (100)
Totalb	27/41 (65.9)	26/41 (63.4)	41/41 (100)	41/41 (100)

^aFg, fibrinogen; Fn, fibronectin.

^bDue to a high capsule production under the experimental settings, human isolate C29 could not be investigated, because no pellet was obtained after centrifugation.

The percentage of S. equi subsp. zooepidemicus isolates showing fibrinogen binding in this Western blot analysis was significantly higher in the human than in the equine isolates (Table 1). The difference was significant for equine fibrinogen (79.2% versus 47.1%; Fisher’s exact test, P = 0.0476) and highly significant for human fibrinogen (83.3% versus 35.3%; Fisher’s exact test, P = 0.0028). In contrast, all analyzed isolates bound equine and human fibronectins. These results suggest that binding of human fibrinogen in larger amounts is a very common phenotype of zoonotic S. equi subsp. zooepidemicus isolates but much less common for S. equi subsp. zooepidemicus isolates of equine origin.

Quantification of fibrinogen binding by flow cytometry.

Western blot analysis of the acidic eluates of the bacterial surface after incubation in plasma (plasma absorption assay) indicated that S. equi subsp. zooepidemicus strains display large differences in the amounts of bound fibrinogen. To verify these results, fibrinogen binding of S. equi subsp. zooepidemicus isolates (n = 41) was quantified by flow cytometry measurements using FITC-labeled equine and human fibrinogens. Results of one representative experiment are shown in Fig. 7. Human isolates bound significantly larger amounts of human fibrinogen (n = 24, average geometric mean [geomean] of 236.5) than equine isolates (n = 17, average geomean of 48.3) (Fig. 7B). The average values calculated for equine fibrinogen did not differ significantly between equine (average geomean of 99.5) and human (average geomean of 115.0) isolates (Fig. 7A). Thus, the binding of large amounts of human fibrinogen is a common phenotype in human but not in equine isolates of S. equi subsp. zooepidemicus.

FIG 7.

S. equi subsp. zooepidemicus of human origin bound larger amounts of human fibrinogen than S. equi subsp. zooepidemicus of equine origin. Equine and human isolates were analyzed for the binding of equine (A) and human (B) FITC-labeled fibrinogen by flow cytometry. The binding of equine and human FITC-labeled fibrinogens was analyzed in correlation to the identified SzM cluster (C and D). The amount of bound fibrinogen is shown as the geomean of fluorescence intensity. One representative experiment of three is shown.

We analyzed further if fibrinogen binding is associated with distinct SzM clusters. No associations were observed for equine fibrinogen, except that the average amount of equine fibrinogen bound by SzM cluster I isolates was significantly higher than the average amount bound by SzM cluster II isolates (Fig. 7C). In contrast, the average amounts of human fibrinogen bound by SzM cluster I and cluster II isolates were significantly higher than the average amounts bound by SzM cluster III and the remaining isolates (Fig. 7D).

Interestingly, the N-terminal parts of SzM of cluster I and cluster III showed 70% amino acid sequence similarity (74% amino acid sequence similarity of proteins without signal peptide to the LPXTG motif) but differed significantly in the amounts of bound human fibrinogen. Although amino acid sequence similarity of cluster I and cluster II SzM is low (24%), the isolates bound the highest average amounts of human fibrinogen. Thus, the results suggest that SzM has different fibrinogen binding motifs.

DISCUSSION

S. equi subsp. zooepidemicus binds to a number of host plasma proteins through cell surface components. Specifically, binding to immunoglobulin G (20, 21), serum albumin (20), fibronectin (22), collagen (23), and α-macroglobulin (20) has been reported. In the present study, binding of equine and human fibrinogens to the surface of zoonotic S. equi subsp. zooepidemicus isolates is demonstrated. The M-like protein SzM is crucial for recruiting equine fibrinogen to the bacterial surface as shown for two S. equi subsp. zooepidemicus strains. Accordingly, fibrinogen binding of the closely related causative agent of strangles, S. equi subsp. equi, depends on expression of the homologous SeM (24). Furthermore, we have shown that the respective recombinant SzM proteins bind to immobilized fibrinogen, which is in accordance with a previous study (8). Thus, at least some variants of SzM bind soluble as well as immobilized fibrinogen. Importantly, we show for the first time that binding of human fibrinogen is a characteristic phenotype of zoonotic S. equi subsp. zooepidemicus isolates, though our results also indicate large differences in binding of human fibrinogen among these isolates. Binding of equine and human fibrinogens are distinct phenotypes in S. equi subsp. zooepidemicus, though SzM expression is crucial for both.

Binding of fibrinogen is known to be important for resistance against opsonophagocytosis in different S. pyogenes serotypes (25–27). Recruitment of fibrinogen to the bacterial surface via M-proteins reduces deposition of the opsonin C3b in different S. pyogenes serotypes (26, 28–30). The interaction of the M1 protein with fibrinogen is crucial for the high virulence of this S. pyogenes serotype in a murine necrotizing fasciitis model (30). Furthermore, the expression of the M-like protein Mrp mediates bacterial survival in human blood via binding of fibrinogen (26). In S. equi subsp. equi, expression of the M-like protein SeM is important for survival in equine blood (24). Here, we show that for the related pathogen S. equi subsp. zooepidemicus, the survival of szm mutants of strains C2 and C33 is significantly attenuated in human blood, that C2ΔSzM is very efficiently killed in equine blood in contrast to the wt, and that SzM expression is necessary for fibrinogen binding in these strains, at least for the level observed in the wt strains. Noteworthy, flow cytometric analysis of szm mutants suggests some residual binding activity for equine fibrinogen (Fig. 1E). One might speculate that this is due to the expression of SzP, another M-like protein of S. equi subsp. zooepidemicus. The homologous protein SzPSe of S. equi subsp. equi binds fibrinogen and is thought to protect S. equi subsp. equi against phagocytosis and deposition of C3 on the cell surface (31). However, expression of SzP in S. equi subsp. zooepidemicus is obviously not sufficient to complement for loss of functions of SzM in fibrinogen binding and immune evasion in blood.

Infection assays of C2 wt and its isogenic mutant C2ΔSzM in reconstituted equine blood showed a significantly lower proliferation of C2ΔSzM in plasma (fibrinogen source) reconstituted blood than of C2 wt, whereas no significant difference was observed in serum reconstituted blood (essentially no fibrinogen). Thus, it is reasonable to suggest that SzM mediates survival in equine blood through fibrinogen binding.

One might argue that proliferation of S. equi subsp. zooepidemicus strain C2 in human blood (Fig. 4B) is not in accordance with the postulated role of fibrinogen binding in immune evasion, as strain C2 binds comparably small amounts of human fibrinogen (Fig. 1F) and rSzM_C2 has a low affinity for human fibrinogen (Fig. 3F). However, only the zoonotic S. equi subsp. zooepidemicus strain C33, which binds equine and human fibrinogens in comparable amounts (Fig. 1), shows similar survival factors in equine and human blood under the chosen experimental conditions (Fig. 4). Furthermore, the difference between C2 and its isogenic szm mutant is not as pronounced in human blood as it is in equine blood. However, as SzM is a multifunctional protein, this attenuation might also be related to other functions, such as IgG binding in the Fc region. Our results demonstrate that rSzM_C2 and rSzM_C33 bind human IgG-Fc. Noteworthy, an IgG-binding domain was described in SeM of S. equi subsp. equi (24), but Velineni and Timoney (8) did not detect binding of equine IgG to the recombinant SzM protein of S. equi subsp. zooepidemicus strain NC78.

Sequence analysis led to the identification of two SzM clusters (I and II) of this highly diverse protein associated with the binding of large amounts of human fibrinogen. Noteworthy, most of the S. equi subsp. zooepidemicus isolates carrying szm genes of clusters I and II were isolated from humans, indicating a high zoonotic potential of these two clusters. Similar to emm typing of S. pyogenes (which is based on 180 bases of the 5′ region of the gene) only the 5′ region of szm was used for this analysis. SzM sequences of our collection showed a much higher degree of diversity than SzP (results not shown). We speculate that SzM diversity is a result of immunological selection and that these large surface proteins carry B cell epitopes and are accessible to antibodies. Accordingly, Velineni and Timoney (8) found SzM of strain NC78 to be a protective antigen using a murine model.

S. equi subsp. zooepidemicus infections in humans are very often invasive and associated with a severe clinical course. Infective endocarditis is an example of severe S. equi subsp. zooepidemicus infection in humans (5, 7, 9, 32–34). Binding of fibrinogen and fibronectin to the surfaces of Gram-positive bacteria is considered to be important for the pathogenesis of endocarditis (35). As the zoonotic S. equi subsp. zooepidemicus strains bound larger amounts of fibrinogen than the other investigated S. equi subsp. zooepidemicus strains, we speculate that this characteristic phenotype might be important for the high frequency of endocarditis following S. equi subsp. zooepidemicus bacteremia in humans.

Few studies have analyzed SzM of S. equi subsp. zooepidemicus isolated from diverse clinical manifestations or host species (8, 16, 18, 36). Two SzM sequences of S. equi subsp. zooepidemicus isolates from humans were available and included in the phylogenetic analysis (Fig. 5). Isolate MGCS10565 caused a severe epidemic of human nephritis in Brazil (16). Interestingly, SzM_MGCS10565 grouped into SzM cluster III, which is mainly composed of human isolates. The SzM of an American isolate which caused a recurrent bacteremia in an infant (SzM_infant) (36) grouped in SzM cluster II, composed of only human isolates. Thus, distinct SzM variants are associated with infections in humans. As our SzM analysis included isolates from different countries, this association might be independent of the geographic region. However, the number of analyzed SzM proteins in this study is limited, and further studies are warranted. The clustering of zoonotic strains is associated with distinct phenotypes of these strains, namely, the binding of human fibrinogen. S. equi subsp. zooepidemicus strains of the zoonotic SzM clusters I, II, and III showed large amounts of human fibrinogen on the bacterial surface after incubation in plasma. These results indicate that binding of fibrinogen mediated by specific SzM types is important for invasive S. equi subsp. zooepidemicus infections in humans.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The analyzed Streptococcus equi collection comprised two S. equi subsp. equi isolates (C1 and C26 from Leipzig and Aachen, respectively) and 42 S. equi subsp. zooepidemicus isolates (see Table S2 in the supplemental material). Twenty-five of the S. equi subsp. zooepidemicus isolates were isolated from humans and 17 were of equine origin. The equine isolates were collected in the Clinic for Horses of the University of Leipzig between 2015 and 2016. The human isolates were from Germany (8 isolates, collected between 2003 and 2014, National Reference Laboratory on Streptococcal Diseases, Aachen, Germany) and from Sweden (17 isolates, collected between 2003 and 2015, Lund, Sweden) (12). S. equi was grown at 37°C without agitation in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY), unless otherwise stated. Identification of bacteria, including subspecies differentiation, was done by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis and biochemical analysis (fermentation of sorbitol and trehalose).

pGHost-transformed Escherichia coli TG1 repA⁺ strains were cultured in Luria Bertani (LB) broth containing erythromycin at 150 μg/ml. Transformed S. equi subsp. zooepidemicus strains carrying recombinant plasmids were grown on THY agar containing erythromycin at 0.5 μg/ml or in THY containing erythromycin at 1.0 μg/ml. E. coli strains XL1 blue and TG1 repA⁺ transformed with pQE30 were cultured in LB broth or on LB agar containing ampicillin at 100 μg/ml at 37°C.

Standard PCR and sequencing of szm.

Genomic DNA was extracted from overnight cultures with the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s instructions. PCR was performed using 0.1 μM each forward and reverse primer, 0.2 mM (each) deoxynucleoside triphosphate (dNTP), 1× OneTaq Quickload reaction buffer, and 1.25 units of OneTaq DNA polymerase (New England BioLabs) per 50-μl reaction volume. The sequences of all oligonucleotide primers used in this study are provided in Table S1 in the supplemental material.

Universal primers SeM/SzM_fwd and SeM/SzM_rev were used to amplify and sequence sem and szm. These primers were designed on the basis of the S. equi subsp. equi 4047 genome. Primer SeM/SzM_fwd is complementary to nucleotides −15 to +10 of the sem gene. Primer SeM/SzM_rev is complementary to nucleotides +1616 to +1596. This region includes the TAA stop codon (1602 to 1604) of sem.

For seven isolates, it was not possible to obtain an amplification product with the above-mentioned primer pair. Thus, the primer pair SzM_down_fwd and SzM_up_rev was used instead to amplify the szm region. These primers were also designed on the basis of the S. equi subsp. equi 4047 genome. Primer SzM_down_fwd is complementary to nucleotides −515 to−494. Primer SzM_up_rev is complementary to nucleotides +2119 to +2088. This region is approximately 480 bp upstream of the stop codon of sem.

Specific primers, designed on the basis of the sequencing results of the szm region PCR amplicons (SzM_C28_fwd and SzM_C13_fwd, complementary to nucleotides around the start codon), were used to analyze the remaining szm sequences. For isolates C19 and C20, it was not possible to obtain valid sequences for szm.

The amplification conditions were as follows: initial denaturation at 94°C for 30 s, 27 cycles of denaturation at 94°C for 15 s, annealing at primer-specific temperatures (Table S1) for 30 s, and extension at 68°C for 1 min per kb of gene length. The cycle reaction was followed by a final extension phase at 68°C for 5 min. PCR products were analyzed by agarose gel electrophoresis and purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) before subjecting to sequencing by Seqlab (Göttingen, Germany).

Analysis of sequences.

DNA analysis and in silico translation of nucleotide sequences into amino acid sequences were performed with BioEdit. Isolate C68 contained a frameshift mutation within its szm gene and was excluded from further sequence analysis. Signal peptide sequences were identified by SignalP (37), and phylogenetic analysis was conducted at the webpage www.phylogeny.fr (38).

In-frame deletion mutagenesis.

To generate szm-deficient S. equi subsp. zooepidemicus mutants by allelic replacement, copies of the S. equi subsp. zooepidemicus C2 and C33 szm genes containing in-frame deletions were constructed. PCR using Phusion polymerase, S. equi subsp. zooepidemicus C2 and C33 chromosomal DNA, and the universal primer pair SzM_down_fwd_IVA and SzM_up_rev_IVA (Table S1) generated amplification products of the szm genes, including approximately 500 bp upstream and downstream of the genes. These fragments were introduced by IVA cloning (39) into the inverse amplified vector pGHost (primers pGhost_fwd_IVA and pGhost_rev_IVA, excluding the ISS1 region) (Table S1) and transformed into E. coli TG1 repA⁺.

The resulting recombinant plasmids pGHost_SzM_C2 and pGHost_SzM_C33 were used as the templates in inverse PCR amplifications with primer pairs SzM_C2_down_rev_NcoI plus SzM_C2_up_fwd_NcoI and SzM_C33_down_rev_NcoI plus SzM_C33_up_fwd_NcoI (Table S1), respectively, to delete the szm genes. Amplification products were digested with NcoI, ligated, and transformed into E. coli TG1 repA⁺ to produce the recombinant plasmids pGHost_ΔSzM_C2 and pGHost_ΔSzM_C33. By inverse PCR, nucleotides 163 to 1596 of szm of strain C2 were deleted, resulting in pGHost_ΔSzM_C2; thus, the central 1,433 bp of szm are lacking. In pGHost_ΔSzM_C33, nucleotides 151 to 1117 of szm of strain C33 were deleted by inverse PCR; thus, the central 966 bp of szm are lacking. pGHost is a replication thermosensitive plasmid that replicates at 28°C but is lost above 37°C (40). While E. coli TG1 repA⁺ allows stable replication of the plasmid pGHost at 37°C, Streptococcus equi will lose the plasmid at 37°C (40).

Allelic-replacement mutagenesis.

Transformations of S. equi subsp. zooepidemicus C2 with the plasmid pGHost_ΔSzM_C2 and S. equi subsp. zooepidemicus C33 with the plasmid pGHost_ΔSzM_C33 were achieved by electroporation. Briefly, overnight cultures of S. equi subsp. zooepidemicus C2 and C33 were diluted 20-fold in 200 ml THY and grown to an optical density at 600 nm (OD₆₀₀) of 0.125. Bacterial cells were harvested by centrifugation and washed twice with ice-cold 0.5 M sucrose. The final wash was with ice-cold 0.5 M sucrose-15% glycerol. The bacterial pellets were resuspended in ice-cold 0.5 M sucrose-15% glycerol, and 100-μl aliquots of the competent cells were used in electroporation reactions. The transformations were performed with 1 to 5 μg plasmid DNA using a Gene Pulser Xcell (Bio-Rad) with pulse settings of 2.5 kV/cm, 200 Ω, and 25 μF. One milliliter ice-cold THY was added to the transformed bacteria. After phenotypic expression through incubation at 37°C for 1 h, transformants were selected by plating serial dilutions on THY agar containing 0.5 μg/ml erythromycin and incubating overnight at 28°C (the permissive temperature) to allow plasmid replication. Allelic replacement was achieved essentially as described previously (41). Briefly, to replace the wild-type szm with the respective in-frame-deleted alleles, transformants containing either pGHost_ΔSzM_C2 or pGHost_ΔSzM_C33 were subjected to two rounds of homologous recombination. The first recombination event, leading to the integration of pGHost_ΔSzM_C2 and pGHost_ΔSzM_C33 into the chromosomes of strain C2 and C33, respectively, was achieved by growing transformants on THY agar containing 0.5 μg/ml erythromycin and incubating overnight at 37°C. Colonies were then inoculated in THY containing 1 μg/ml erythromycin and grown at 37°C. Dilutions (1:50) of overnight cultures were incubated in THY at 28°C overnight. Incubation at the permissive temperature allows plasmid replication and facilitates the second recombination event. Serial dilutions were plated on blood agar plates and grown at 37°C to subsequently screen for bacteria that lost the excised plasmid. Putative mutant colonies were subcultured on blood agar plates and THY agar containing 0.5 μg/ml erythromycin to confirm their erythromycin sensitivity. The mutants were screened by PCR using the primers SzM_down_fwd and SzM_up_rev. Predicted deletions were confirmed by DNA sequencing. Mutant strains were designated C2ΔSzM and C33ΔSzM.

Cloning of szm into pQE30.

Cloning of szm genes into the expression plasmid pQE30 was achieved by standard DNA manipulations (42) or by IVA cloning (39). In detail, szm sequences, coding for the mature SzM protein (excluding the signal peptide and the region downstream of the carboxy-terminal LPXTG anchor motif), were amplified and cloned into pQE30.

All PCRs were conducted using Phusion polymerase (New England BioLabs). Briefly, chromosomal DNA of S. equi subsp. zooepidemicus isolate C33 served as the template for PCR with primer pair C33_BamHI_fwd plus SzM_KpnI_rev. The resulting PCR product as well as the plasmid pQE30 was digested with BamHI and KpnI and ligated and transformed into E. coli XL1 blue, resulting in the recombinant plasmid pQE30_SzM_C33 encoding the mature 347-aa SzM_C33 protein.

The szm gene of S. equi subsp. zooepidemicus isolate C2 was introduced into pQE30 by IVA cloning using separate PCR amplification products. Briefly, szm of isolate C2 was amplified with primer pair SzM_C2_fwd_IVA_pQE30 plus SzM_rev_IVA_pQE30, and the plasmid pQE30 was amplified with primer pair pQE30_IVA_fwd plus pQE30_IVA_rev. PCR products were mixed and transformed into E. coli TG1 repA⁺, because IVA cloning in E. coli XL1 blue failed. Afterwards, the resulting plasmid pQE30_SzM_C2 encoding the mature 503-aa SzM_C2 protein was transformed into E. coli XL1.

For cloning of the N terminus of SzM of strain C33 (first 216 aa of the mature protein), plasmid pQE30_SzM_C33 served as the template and was amplified with primers pQE30_IVA_fwd and SzM_C33_Nterm_IVA_pQE30_rev. The PCR product was transformed into E. coli TG1 repA⁺ and afterwards subcloned into E. coli XL1.

Expression and purification of recombinant His-tagged proteins.

E. coli isolates harboring the respective plasmids were grown in LB broth with ampicillin to an OD₆₀₀ of 0.5. Once the OD₆₀₀ was reached, isopropyl-β-d-thiogalactopyranoside (IPTG) (AC121; VWR) at a final concentration of 1 mM was added, and the cultures were incubated at 37°C for 4 h under constant shaking. Following centrifugation, bacteria were resuspended in LEW buffer (catalog number 745120.25, Protino Ni-TED; Macherey-Nagel) and lysed by passing multiple times through a French press. Recombinant proteins were then purified under native conditions via Ni-TED columns as recommended by the manufacturer (catalog number 745120.25, Protino Ni-TED; Macherey-Nagel) and dialyzed against phosphate-buffered saline using dialysis membranes with a molecular weight cutoff of 6,000 to 8,000 Da (catalog number E660.1, ZelluTrans; Carl Roth).

ELISAs of recombinant SzM–IgG-Fc and -fibrinogen interactions.

For analysis of the rSzM–IgG-Fc interaction, Corning 96-well Costar plates (clear flat-bottom polystyrene high-bind microplate, product number 9018) were coated with doubling dilution series of rSzM_C2, rSzM_C33, and negative-control rMRP (muramidase-released protein of Streptococcus suis), leading to a range of 0.03125 to 2.0 μg/well in triplicates at 4°C overnight. Wells were washed three times with 200 μl TBS-T (20 mM Tris, 154 mM NaCl, 0.05% [vol/vol] Tween 20) followed by a blocking step with 200 μl 5% skim milk in TBS-T at 37°C for 1 h. After washing three times with 200 μl TBS-T, wells were incubated with 50 μl 1:10,000 human IgG-Fc conjugated with HRP (Jackson; 009-030-008) in TBS-T at 37°C for 1 h. After washing four times with 200 μl TBS-T, 100 μl 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) solution (0.1 M citric acid in double distilled water [ddH₂O], pH 4.35, 500 μg/ml ABTS) with H₂O₂ was added to the wells and incubated at 37°C for 30 min. Color development was measured at OD₄₀₅. Three separate experiments were performed.

For analysis of the rSzM-fibrinogen interaction, 96-well Costar plates were coated with doubling dilution series of equine fibrinogen (see “Purification of equine fibrinogen,” below) and human fibrinogen (F3879; Sigma) (4 μg/well to 0.03125 μg/well, in triplicates) at 4°C overnight. Wells were washed three times with 200 μl TBS-T followed by a blocking step with 200 μl 5% skim milk in TBS-T at 37°C for 1 h. After washing three times with 200 μl TBS-T, wells were incubated with 100 μl recombinant SzM in various concentrations (1.0 μg/ml, 0.5 μg/ml, 0.25 μg/ml, and 0.1 μg/ml, in triplicates) in TBS-T at 37°C for 1 h. After washing three times with 200 μl TBS-T, wells were incubated with 50 μl 1:10,000 human IgG-Fc conjugated with HRP (009-030-008; Jackson) in TBS-T at 37°C for 1 h. After washing four times with 200 μl TBS-T, 100 μl ABTS solution with H₂O₂ was added to the wells and incubated at 37°C for 30 min. Color development was measured at OD₄₀₅. Three independent experiments were performed.

As a second method for the analysis of the rSzM-fibrinogen interaction, biotinylated rSzM was used. Biotinylation of rSzM was conducted with EZ-Link Sulfo-NHS-LC-Biotin (A39257; Thermo Fisher) according to the manufacturer’s instructions. Ninety-six-well Costar plates were coated with doubling dilution series of equine or human fibrinogen (1 μg/well to 0.01563 μg/well, in triplicates) at 4°C overnight. Wells were washed three times with 200 μl TBS-T followed by a blocking step with 200 μl 2.5% essentially globulin free bovine serum albumin (BSA) (A7030; Sigma) in TBS-T at 37°C for 1 h. After washing three times with 200 μl TBS-T, wells were incubated with 100 μl biotinylated recombinant SzM (0.5 μg/ml of rSzM_C2-biotin or rSzM_C33-biotin, in triplicates) in TBS-T with 0.25% BSA at 37°C for 1 h. After washing three times with 200 μl TBS-T, wells were incubated with 100 μl of a 1:10,000 dilution of streptavidin conjugated with HRP (catalog number 405210; BioLegend) in TBS-T with 0.25% BSA at 37°C for 1 h. After washing four times with 200 μl TBS-T, 100 μl ABTS solution with H₂O₂ was added to the wells and incubated at 37°C for 30 min. Color development was measured at OD₄₀₅. Three independent experiments were performed.

Whole blood bactericidal assay.

Survival of S. equi subsp. zooepidemicus wt and isogenic mutants in blood was analyzed using whole heparinized equine or human blood. Handling of horses and sampling was conducted in strict accordance with the principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes as well as the German Animal Protection Law. The withdrawal of blood was part of an animal experiment which was approved by the Committee on Animal Experiments of the Lower Saxonian State Office for Consumer Protection and Food Safety under the number TVV 45/18. Human blood samples were collected from six healthy donors in agreement with the local ethical board (approved by the Ethic Committee of Hannover Medical School [MHH], Hannover, Germany) registered under number 3295-2016.

Briefly, 0.5 ml equine or human whole blood was mixed with 1 × 10⁵ CFU of wt or mutants. The mixture was incubated on a rotator at 37°C for 120 min. Survival factors were determined by dividing the number of CFU after a 120-min incubation period at 37°C by the number of CFU at t = 0 min (CFU were determined by plating of serial dilutions on blood agar plates). Each assay included 8 blood samples from different horses. Seven of eight horses used for withdrawal of blood were identical for the assays with C2/C2ΔSzM and C33/C33ΔSzM.

Reconstituted blood bactericidal assay.

Additionally, the survival of S. equi subsp. zooepidemicus isolate C2 and its isogenic mutant C2ΔSzM in correlation with fibrinogen was analyzed using a reconstituted equine blood bactericidal assay. Thus, 7 ml whole heparinized blood was centrifuged at 3,000 × g for 5 min to separate plasma and blood cells. Plasma was taken off and stored at room temperature. Blood cells were washed four times with 40 ml phosphate-buffered saline (PBS) and finally supplemented with PBS to the original blood volume. Five hundred microliters of the cell suspension in PBS was transferred to 1.5-ml tubes and centrifuged at 3,000 × g for 5 min. Two hundred fifty microliters of supernatant was removed, and blood cells were resuspended in 250 μl endogenous freshly drawn serum and plasma. Ex vivo infection of the reconstituted blood was performed as described for the whole blood bactericidal assay. Each assay included blood samples from 4 different horses.

Plasma absorption assay with Western blot.

S. equi subsp. zooepidemicus was grown in THY to an OD₆₀₀ of 0.5. After washing the bacteria with PBS, the sample was adjusted to an OD₆₀₀ of 0.5, and each 10 ml of the same bacterial suspension was incubated with 100 μl equine and human plasma at 37°C for 1 h. After removal of unbound proteins by washing two times with 10 ml of PBS, bound proteins were eluted with 160 μl 100 mM glycine-HCl (pH 2) at room temperature (RT) for 15 min. Solid material was removed by centrifugation (5 min, 10,000 × g). Supernatant was mixed with SDS-PAGE sample buffer and subjected to SDS-PAGE using stacking and separation gels of 4% and 12% acrylamide, respectively. Following electrophoresis, proteins were visualized by staining with InstantBlue (Expedeon). For immunoblots, proteins were transferred to nitrocellulose. Membranes were blocked with 5% (wt/vol) skim milk powder in TBS-T and probed with goat anti-human fibrinogen antibody (cross-reacts with equine fibrinogen, F8512; Sigma) and rabbit anti-human fibronectin antibody (cross-reacts with equine fibronectin, F3648; Sigma). After washing three times with TBS-T, membranes were probed with a rabbit IgG anti-goat IgG HRP-conjugated antibody (305-035-003; Dianova) and goat IgG anti-rabbit IgG HRP-conjugated antibody (111-035-008; Dianova), respectively. Equine and human IgGs were detected with goat IgG anti-horse IgG HRP (108-035-003; Dianova) and goat IgG anti-human IgG HRP (109-035-088; Dianova), respectively. After washing three times with TBS-T, peroxidase activity was detected by chemiluminescence using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific). The marker bands were detected via the visible imaging tool and the Western blot bands through the chemiluminescence application of the Fusion SL system. The marker bands are positioned by the Fusion SL system automatically as shown in the joined figure.

Western blot of human IgG-Fc fragment binding.

Recombinant proteins rSzM_C2, rSzM_C33, and rMRP were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with 5% (wt/vol) skim milk powder in TBS-T and probed with (i) peroxidase-conjugated human IgG-Fc fragment (009-030-008; Jackson) and (ii) mouse IgG2b anti His-tag antibody (MA1-21315; Thermo Fisher) and goat IgG anti mouse IgG HRP-conjugated antibody (150-035-071; Dianova). After washing three times with TBS-T, peroxidase activity was detected by chemiluminescence using SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific).

Purification of equine fibrinogen.

Equine citrate plasma was thawed overnight on ice and centrifuged two times at 16,300 × g at 4°C for 8 min to remove cold-insoluble fibronectin. Supernatant was mixed 1:2 with 10% polyethylene glycol 8000 (PEG 8000) in ddH₂O and incubated at RT for 15 min. The pellet, obtained by centrifugation at 16,300 × g at RT for 15 min, was resuspended in PBS. Fibrinogen was precipitated by the addition of 8% ethanol ([vol/vol] final concentration) and incubated on ice for 1 h. The pellet, obtained by centrifugation at 16,300 × g at 4°C for 15 min, was dried and resuspended in PBS containing 5 mM EDTA.

Bacterial binding of FITC-labeled fibrinogen.

Purified equine fibrinogen and human fibrinogen (F3879; Sigma) were labeled with fluorescein isothiocyanate isomer I (FITC) (21590; Serva) as described by Sigma. FITC-labeled fibrinogen was dialyzed against PBS containing 5 mM EDTA.

Bacterial binding of FITC-labeled fibrinogen was performed as follows. S. equi subsp. zooepidemicus was prepared as described under “Plasma absorption assay with Western blot,” above. The bacterial suspension was incubated with FITC-labeled equine and human fibrinogens at 37°C for 1 h. After washing the bacteria to remove unbound proteins, the bacterial pellet was resuspended in 2% paraformaldehyde in PBS. Fluorescence intensity was measured by flow cytometry after excitation with 488 nm laser with the Becton, Dickinson FACSCalibur at 520 nm after gating for intact bacteria in the forward scatter/side scatter (FSC/SSC) plot. The data were analyzed with FlowJo_V10 software. Representative plots are shown in Fig. S1.

Accession number(s).

Nucleotide sequences of szm were deposited at GenBank under the accession numbers MH286971.1 to MH286999.1.

We dedicate this work to the memory of our friend and colleague Gursharan Singh Chhatwal.