The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number (n = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.

Enteroaggregative Escherichia coli infection is initiated by colonization of the small and large bowel mucosal surfaces by aggregative adherence. This is followed by biofilm formation, induction of an inflammatory response, and release of toxins (1). The precise mechanisms of pathogenesis are still not fully understood, but a combination of several factors such as adhesins and toxins are described to contribute to disease (4,5). However, none of these factors are conserved in all EAEC strains and a number of similar factors are found in other E. coli pathotypes, suggesting that EAEC pathogenesis does not depend on one particular protein, but is probably based on a combination of several virulence factors (2,4).
Enteroaggregative Escherichia coli strains can be recovered from stool samples of apparently healthy individuals and despite studies finding strains associated with diarrhea, some studies have failed to show significant association between EAEC and disease (6)(7)(8). This suggests that host factors are involved in manifestations of gastrointestinal disease and fur ther investigations could be crucial for the understanding of EAEC pathogenesis.
The complement system is a complex surveillance system involved in innate immune protection against pathogens. It faci litates opsonophagocytosis of pathogens, induces inflammatory responses, and can lead to bacterial lysis upon activation. Acti vation can occur via three pathways: the lectin, the classical, and the alternative pathway. The complement system is primarily regarded to be of importance for systemic immune protection. But, also local production of complement components is recog nized as being important as exudation of complement from the circulation during inflammation appears to be important for local innate immune protection (9).
In the lectin pathway, mannosebinding lectin (MBL) and ficolin1, 2 and 3 are patternrecognition molecules (PRMs) involved in initiation of complement activation (10). Recently, two other molecules collectin10 (CL10 or CLL1) and collec tin11 (CL11 or CLK1) have to some degree been shown to mediate complement activation (11,12). They interact with pathogenassociated molecular patterns on the surface of micro bial pathogens and upon recognition activate the lectin pathway with help from lectin pathwayassociated serine proteases termed MASPs (13). The MASPs cleave C4 and C2 leading to the formation of the C3 convertase (C4b2a). The C3 convertase cleaves C3 into anaphylatoxin C3a and the strong opsonizing factor C3b. Activation through the classical pathway depends on antibody-antigen recognition, which then binds the PRM C1q and leads to cleavage of C4 and C2 by associated proteases C1r/C1s and to deposition of C3b. The alternative pathway is activated spontaneously by hydrolysis of C3, this allows binding of the factor B, which is then cleaved by factor D, forming the C3 convertase of the alternative pathway (C3bBb). The alternative pathway works like an amplification loop for C3b formation and as C3b level rises the C5 convertase is formed (C4b2aC3b/ C3bBb3b) initiating formation of the terminal lytic C5b9 mem brane attack complex (MAC) (14).
The involvement of complement in EAEC pathogenesis is unresolved, and though it has previously been shown that ficolin2 was able to recognize EAEC (15) the importance of the lectin pathway is yet unknown. Thus, we hypothesized that the lectin pathway molecules MBL, ficolin1, 2, and 3 could be involved in recognition and thus complement dependent protec tion of EAEC bacteria.

Bacterial strains
Four prototype EAEC strains, producing aggregative adherence fimbriae (AAF) I-IV, were investigated for binding of lectin pathway recognition molecules MBL, ficolin1, ficolin2, and ficolin3. The strains have been described previously (16). In addition, 56 EAEC strains isolated from stool samples of Danish adults suffering from diarrhea, at the diagnostic laboratory at Statens Serum Institut, were randomly selected. Stock cultures were frozen at −80°C in LuriaBertani broth (LB, SigmaAldrich) containing 10% (vol/vol) glycerol. Bacteria were cultivated in Dulbecco's modified eagle medium containing 4.5 g/l dGlucose (DMEMHG, Gibco™) overnight with shak ing at 37°C until reaching an optical density (OD600 nm) of 1.8, corresponding to a bacterial concentration of approximately 5 × 10 8 cells/ml.

consumption assay
We screened four prototypic EAEC strains for binding of recombinant proteins, and 56 clinical EAEC isolates for bind ing of serum ficolin2. The overnight bacterial cultures were centrifuged at 5,000 × g for 5 min and washed three times in phosphatebuffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 2 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4). The cell pellet was resuspended in BarbitalTween buffer (BarbT, 5 mM barbital sodium, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 0.05% Tween 20, pH 7.5) [barbital buffer previously used by Rosbjerg et al. (18); Hummelshøj et al. (19)] and incubated with 10% normal human serum (NHS) pool originating from four healthy donors, for 1 h at 4°C, or inhouse recombinant proteins for 2 h at 37°C, endoverend. Recombinant proteins were used in the follow ing concentrations: recombinant ficolin1 (rficolin1) 2 µg/ml, rficolin2 0.5 µg/ml, rficolin3 0.5 µg/ml, and recombinant MBL (rMBL) 0.5 µg/ml. After centrifugation (5,000 × g, 5 min) the supernatant was transferred to quantification assays (described below) where the level of consumption was evaluated by compar ing the amount of remaining protein in the supernatant with a control sample containing no bacteria. Serum ficolin2 screen ings were performed using NAcetyldglucosamineAgarose (GlcNAc) beads (SigmaAldrich) as a positive binding control matrix.

elisa-Determination of Unbound Protein Fraction
Maxisorp polystyrene microtiter plates (Thermo Scientific) were coated with 5 µg/ml acetylated bovine serum albumin or 10 µg/ml mannan in PBS, overnight at 4°C. Plates were washed and blocked in BarbT before adding the supernatants (from the consumption assay) in serial dilutions and incubating over night at 4°C. Plates were washed in BarbT, and detection was performed using the following primary monoclonal antibodies (mAb) in a concentration of 2 µg/ml at 20°C: antificolin1 mAb (HP9039, Hycult biotech), antificolin2 mAb clone FCN219 (20), antificolin3 clone FCN334 (21), and HYB 13111 (Bioporto Diagnostics) for MBL detection. Plates were incubated 2 h at room temperature, shaking. Plates were washed and HRPconjugated rabbit antimouse polyclonal antibody (P0260, Dako) (1:1,500) was added for 45 min at room tempera ture, shaking. Plates were thoroughly washed with BarbT and subsequently developed for 20 min with tetramethylbenzidine One (TMB ONE, KemEnTec Diagnostics). The reaction was stopped with 0.2 M sulfuric acid (H2SO4) and OD was measured at 450 nm.

Western Blot-Detection of Bound Proteins
The bacterial cell pellets (from the consumption assay) were washed thoroughly in BarbT and analyzed by western blotting. Bacteria were lysed with LDS sample buffer (Invitrogen) and the content was run on a 4-12% bisTris polyacrylamide gel (Invitrogen). Rficolin2 (0.25 µg) was used as a loading control. The separated proteins were blotted onto polyvinylidene difluoride membranes (GE Healthcare) and the membranes were probed with 0.5 µg/ml antificolin1 mAb FCN106 (cross reacting with ficolin2) overnight at 4°C (22). After washing, the membranes were incubated with rabbit antimouseHRP (1:10,000) (P0260, Dako) for 1 h at room temperature, shaking. Membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).

Flow cytometry-Detection of complement activation
Prior to assessing binding and activation with flow cytometry, strain C32217 was fixed in formalin. An overnight culture of C32217 was washed in PBS and the concentration was deter mined by a colonyforming unit (CFU) count. Bacteria were fixed in 4% formalin for 40 min, washed in PBS, and resuspended in 50% ethanol. The stock was kept at −80°C until use.

Microscopy
Ficolin2 binding in the presence and absence of the ficolin2 inhibitor FCN212 was assessed by microscopy. The residual EAEC cells from flow cytometry was placed on slides by cyto spin (centrifugation for 5 min at 300 × g) and mounted with ProLong Diamond Antifade Mountant (P36965, Life Technolo gies). Microscopy was performed using a Zeiss Axio Observer through a X63/1.40 oil DIC PlanApochromat objective. Imaging conditions were kept constant when acquiring images to be compared.

eaec serum resistance
An overnight bacterial culture of C32217 was diluted to an OD = 0.5 at 600 nm in PBS. 10% NHS were incubated in buffer containing the following inhibitors: FCN212 (5 µg/ml), antiFD (5 µg/ml), C1q85 (5 µg/ml) or mock inhibitor mAb Ciona (a mAb raised against an MBL homolog in Ciona intestinalis). In addition, we used a specific peptide inhibitor of C3 activa tion, compstatin (CP40, 6 µM, a kind gift from professor John Lambris, Philadelphia, PA, USA), and C5 inhibitor eculizumab (50 µg/ml, Soliris, Alexion Pharmaceuticals). Heatinactivated NHS (IHS) (56°C, 30 min) was included as a negative control of complement activation. The baseline consisted of bacteria incubated only with buffer to assess the number of viable cells in the initial inoculum.
All tubes were preincubated at 37°C for 30 min to let the inhibitors work and to start the test approximately at human blood temperature. Then, 20 µl of the inoculum was added to each tube and the sample was homogenized. The samples were incubated at 37°C and 20 µl was spotted in a serial dilution of 10 −1 to 10 −6 on LB agar plates and incubated at 37°C overnight before CFU was assessed. The control sample with viable cells of the initial inoculum was diluted and plated at 0 min incubation.

Binding of rMBl and Ficolins to Prototypic eaec strains
Four prototypic EAEC strains were screened in a consump tion assay for binding of rMBL, rficolin1, 2, and 3. Neither rficolin1 nor rficolin3 appeared to bind to any of the strains (Figures 1B,D), whereas rMBL showed binding to two of the strains (JM221 and 042) ( Figure 1A). The strongest binding, however, was observed for rficolin2, which displayed high binding to two strains (55989 and C101000) and to a lesser degree to strain 042 ( Figure 1C). We confirmed the binding by performing a Western blot on the eluates from the consumption assay (data not shown). We found that rficolin2 displayed highest binding to the four prototypic EAEC strains and, therefore, decided to examine ficolin2 binding to 56 clinical EAEC isolates.
Binding of serum Ficolin-2 to 56 clinical eaec isolates 56 clinical EAEC isolates obtained from adult patients suffer ing from EAECrelated diarrhea were screened for binding of serum ficolin2 and the unbound fraction of serum ficolin2 was measured. Five (8.9%) of the 56 isolates appeared to bind serum ficolin2 and especially one isolate, C32217, showed very strong binding (Figure 2). We furthermore verified that serum ficolin2 was bound to isolate C32217 by performing a Western blot on the bacterial pellet for C32217 and two isolates that were negative for ficolin2 binding according to the ELISA. The Western blot showed strong monomeric and oligomeric ficolin2 structures to be associated with C32217. The two isolates, E31065539 and H57553, which were nega tive for ficolin2 binding in the ELISA displayed low levels of oligomeric ficolin2 binding, but no monomeric bands were observed (Figure 3).
Since isolate C32217 displayed high binding of ficolin2, we decided to use this isolate as a model for further investigation of EAECassociated complement binding and activation.

calcium Dependency of Ficolin-2 Binding
We examined whether ficolin2 binding to isolate C32217 was calcium dependent by preincubating NHS with 10 mM EDTA. Figure 4 shows the mean fluorescence intensities (MFI) when detecting ficolin2 binding in flow cytometry. Ficolin2 bind ing was not reduced by EDTA providing evidence that that the binding was calcium independent. In fact, it seemed that EDTA

complement activation by Ficolin-2 Through the lectin Pathway
The contribution of ficolin2 to lectin pathway complement activation was assessed by introducing FCN212, a ficolin2 inhibitory mAb. Figures 5A,B shows inhibition of ficolin2 binding to the bacterial strain C32217 in flow cytometry and fluorescence microscopy, respectively, when applying the inhibitor. Both techniques showed that the binding was reduced by the employed ficolin2 inhibitor. In flow cyto metry, the inhibition was significantly reduced both when comparing to 10% NHS and to a mock ficolin2 inhibitor (p < 0.0001).
The effect of ficolin2 binding on complement activation was examined by looking at the deposition of C4b, C3b, and   MAC in the presence and absence of the ficolin2 inhibitor. C4b deposition was significantly reduced by the inhibitor when comparing to both 10% NHS and the mock inhibitor (p = 0.0008 and 0.0026, respectively). The same tendencies were observed for MAC deposition when comparing to 10% NHS and the mock inhibitor (p = 0.0189 and 0.0134, respectively), sug gesting an involvement of ficolin2 and the lectin pathway in complement activation (Figures 6A,C). We observed a small significant reduction in C3b deposition when comparing to 10% NHS (p = 0.0495), but were unable to detect a significant reduction when comparing to the mock inhibitor (p = 0.0964), but overall the tendency followed that of C4b and MAC ( Figure 6B). We also looked at MBL binding to see if this was involved in activation via the lectin pathway, but MBL did not bind to C32217 and thus does not contribute to activation (data not shown).

The classical Pathway Does not show involvement in complement activation
Since ficolin2 only seemed to be partially involved in comple ment activation, we investigated whether the remaining activity was initiated via the classical pathway. When introducing an inhibitor of C1q, we saw no significant reduction in the deposi tion of C4b, C3b, or MAC (p > 0.05) (Figures 7A-C, respec tively) and, therefore, the classical pathway does not appear to be involved in the remaining complement activity.
The alternative Pathway contributes to complement activation Next, we tested the involvement of the alternative pathway by applying an inhibitory antibody against factor D before detect ing C3b and MAC deposition (Figure 8). We were unable to detect a significant reduction in C3b levels when introducing the factor D inhibitor alone. However, when combining the factor D inhibitor with the ficolin2 inhibitor, we observed a significant reduction in C3b deposition when comparing both to 10% NHS and to a sample where the ficolin2 inhibitor was exchanged with a mock inhibitor (p = 0.0057 and 0.0002, respectively) ( Figure 8A). The deposition of MAC was highly reduced by the factor D inhibitor alone (10% NHS p = 0.0058 and mock inhibi tor p < 0.0001) and adding the ficolin2 inhibitor led to further reductions when comparing to the mock inhibitor (p = 0.0169) ( Figure 8B). These data suggest that both the lectin pathway and the alternative pathway are important in activation of complement on EAEC strain C32217 and emphasize the importance of the alternative pathway in generation of MAC.

Ficolin-2 and Factor D are involved in serum-Mediated eaec Killing
To further investigate the involvement of ficolin2 and the alternative pathway on bacterial clearance, we performed a serum resistance assay where bacteria were mixed with 10% NHS and the inhibitors of ficolin2, factor D and C1q, as well as the C3targeted complement inhibitor, compstatin, and the terminal complement inhibitor eculizumab that prevents MAC formation. We assessed the change in CFU/ml between 10% NHS and the different parameters. There was a significant reduction when bacteria were grown in 10% NHS compared to the baseline (No NHS) (p = 0.0046). Heatinactivated human serum (IHS) was applied as a control of complement activity and did not only lead to rescue of bacterial growth, but could be interpreted to function as a growth medium for the bacteria. The C1q inhibitor did not rescue the bacteria (p = 0.6904), suggest ing no involvement from the classical pathway in the observed serummediated killing. The ficolin2 inhibitor and the factor D inhibitor each led to significant bacterial rescue (p = 0.0029 and  0.0130, respectively), suggesting involvement of both the lectin and alternative pathway in serummediated killing. Combining the two inhibitors did not appear to further increase the rescue. Targeting C3 and MAC with inhibitors also led to significant rescue of bacterial growth (p = 0.0107 and 0.0038, respectively) (Figure 9).

DiscUssiOn
Enteroaggregative Escherichia coli (EAEC) is a wellknown diar rheagenic pathogen, causing acute and persistent diarrhea in children and adults worldwide. However, the molecular epi demiology of EAEC still remains unclear and several studies have recovered EAEC from stool samples of apparently healthy individuals suggesting the involvement of host factors (2). The lectin pathway of the complement system relies on PRMs to assist in the clearance of microbial intruders. Ficolins are a family of PRMs belonging to the lectin pathway. They bind structures such as Nacetylglucosamine (GlcNAc), Nacetylgalactosamine (GalNAc) and acetylated compounds on target cells (24). Although complement proteins are generally considered in the systemic compartment, recent studies show   production and secretion of complement components in human immune cells, as well as endothelial and epithelial cells (9). This makes the study of interactions between rare or nonsystemic pathogens and complement highly relevant and could potentially give a better understanding of host defense systems, as well as bacterial pathogenesis.
In this study, we assessed the involvement of the lectin comple ment pathway on 56 EAEC strains isolated from patients suffer ing from EAECrelated diarrhea. First, we applied a consumption assay to screen the binding of rMBL and ficolins to prototypic EAEC strains in an ELISA setup. We found that rficolin2 pre sented with the highest binding capacities for the prototype EAEC strains, showing strong binding to prototype strains 55989 and C101000, and to some degree to prototype 042. In a pre vious study no binding of ficolin2 to prototype strain E. coli 042 was detected, but they were using NHS (serum protein) and in a different E. coli growth medium (15). Using a different growth medium for EAEC could potentially change the gene expres sion of the strain and lead to changes in surface presentation. Biofilm formation in some EAEC strains have shown to increase significantly when using a high glucose containing medium as compared to regular LB (25). This emphasizes the importance of the methodology employed when studying bacterial interaction with host factors.
Based on our initial screenings, we focused on the PRM ficolin2. Previous reports have described binding of ficolin2 to Grampositive bacteria, such as group B streptococci, S. pneumoniae, S. pyogenes, and capsulated S. aureus (26)(27)(28), but very few studies have described binding of ficolin2 to Gramnegative bacteria. A study by SahagúnRuiz et al. explored the binding capacity of serum ficolin2 and ficolin3 to Gramnegative bacteria including four EAEC strains. They found that ficolin2 and ficolin3 recognized one EAEC strain (serotype O71), but not the other three. By testing binding to another EAEC serotype O71 they concluded that binding was not related to the bacterial LPS type (15), but did not determine the specific binding factor.
The binding capacity of the 56 clinical EAEC isolates was screened by incubating bacteria with 10% NHS. Ficolin2 bind ing was only detected in five of the 56 EAEC isolates (8.9%). Complement evasion strategies are numerous and include mimicking or recruitment of complement regulators, inhibition of complement proteins, and enzymatic degradation leading to inactivation (29). Both Grampositive and Gramnegative bacteria are capable of evading complement by recruitment of complement regulators, such as factor H and C4bbinding pro tein (C4BP) (30). The E. coli K1, causing neonatal meningitis, utilizes the outer membrane protein A (OmpA) as protection against complementmediated killing, and serum resistance was correlated with the binding of C4bP to OmpA (31,32). Immune evasion has also been reported for EAEC by cleavage of complement proteins by Pic, which is a serine protease present in approximately 50% of clinical EAEC isolates (4,5,33,34). In a study by Abreu et al., they showed that Pic significantly reduced complement activation by cleavage of C3, C3b, C4, and C2, thereby affecting all three complement pathways (35). EAEC is known to cause persistent diarrhea, most likely due to the formation of a resilient biofilm. A study from 2013 showed that biofilm formation worked as an efficient way of evading complement detection (36) and could potentially be a way for EAEC to avoid complement. As part of the present study, we investigated whether ficolin2 binding could be related to the five EAEC characteristic AAF (I-V). By PCR, we characterized the presence of AAFs in the 56 clinical EAEC strains, but we were unable to find a link between binding of ficolin2 and the pres ence of a specific AAF, the strains that bound ficolin2 harbored different AAFs (data not shown). We hypothesize that some bacterial strains are able to change their surface composition in order to avoid ficolin2 binding, thus escaping the initiating effect of complement.
It is important to mention that we did see a difference in detec tion of ficolin2 binding when comparing ELISA with western blot. Two of the strains reported negative for binding in the ELISA, were run on a western blot where we were able to detect low levels of oligomeric binding. This should be tested further to determine the sensitivity of the ELISA.
Before assessing the complement activation potential of EAEC, we determined whether ficolin2 binding was calcium dependent. Xray crystallographic analysis has revealed four different binding sites in the fibrinogenlike domain of ficolin2, enabling binding to a wide variety of ligands (24). While some of these binding sites require the presence of calcium, others can bind calcium independently (10). Using isolate C32217 as a model for high level ficolin2 binding, we could show that ficolin2 binding to the bacteria was calcium independent.
We next showed that ficolin2 binding to EAEC can lead to activation of complement. The activation could be partially inhibited by a ficolin2specific inhibitor. Furthermore, activa tion was independent of the classical pathway, thereby confirm ing the involvement of the lectin pathway of complement. We also show that the alternative pathway contributes to activation and that the amplification loop contributes to a high produc tion of C3b, possibly explaining the need to introduce both a ficolin2 inhibitor and a factor D inhibitor to see a decreasing effect on C3b levels. This observation is compatible with previous studies on fungi showing the importance of alternative path way amplification on lectin pathwayinitiated activation (18).
Finally, we show that ficolin2 and factor D are involved in serummediated killing of EAEC and that this killing is com pletely dependent on the formation of the MAC complex. Although EAEC is not common in sepsis infections, there have been reports of EAECinduced bacteremia (37,38), and this could be due to lack of complement dependent systemic control of the infection.