Interaction of lactoferrin, lysozyme and the complement system in in vitro modeling of innate immune responses

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Introduction

The complement system is a group of proteins located mainly in blood plasma. Complement is an important factor linking humoral and cellular responses, innate and adaptive immunity, and protective and pathological reactions. Its activation via the classical, lectin, or alternative pathways leads to the assembly of C3 and C5 convertases, the production of anaphylatoxins, and the formation of a membrane attack complex (MAC) capable of lysing Gram-negative bacteria [1]. Lactoferrin and lysozyme are classic examples of proteins of innate immunity, and their antimicrobial activity is well described in the literature. Lactoferrin is an iron-binding glycoprotein (~80 kDa) from the transferrin family. Lactoferrin is characterized by multifunctionality, which includes a bacteriostatic effect, direct killing of microbes, as well as regulation of stress and immune responses of the macroorganism [2, 3]. Lysozyme (~ 14.5 kDa) is one of the first discovered immune proteins. Acting as a muramidase, lysozyme hydrolyzes the peptidoglycan of cell walls and, in contrast to complement, is particularly active against Gram-positive bacteria [4]. Lysozyme also attracts attention as an immunomodulatory agent [5]. Furthermore, hen egg white lysozyme (HEWL) is a homologue of human lysozyme (hLZ), which is a model protein for studying lysozyme activity, including modulation of macroorganism immunity [6].

Complement components and innate immunity proteins are co-localized in various body fluids and inflammatory sites. Although the concentrations of lactoferrin and lysozyme in blood plasma are relatively low (~ 1 μg/mL for both proteins), they are among the main components of tear fluid (several mg/mL), milk (several mg/mL for lactoferrin and tens of μg/mL for lysozyme), and among the dominant proteins of neutrophil granules (~ 1–10 μg/10⁶ cells) [7–12]. The level of these proteins can increase significantly in areas of inflammation after the recruitment, activation, and degranulation of neutrophils. Complement components are present in the secretions of various glands and in blood plasma. Like other plasma components, they enter the site of inflammation, where they encounter activated neutrophils. This can be considered a prerequisite for the interaction of complement with innate immunity proteins. Considering that inflammation and immune responses not only protect the body but can also cause damage to cells and tissues, it seems important to study the links between the complement system and antimicrobial peptides as active participants in the immune response. There are, in fact, data in the literature on the interaction of lactoferrin and lysozymes with the complement system. In particular, it has been demonstrated that human lactoferrin inhibits the classical complement pathway and has been identified as the main complement-inhibiting factor in tear fluid [13]. It has been shown that in its apo-form, lactoferrin acts at the C3 convertase level, and the binding of iron and copper ions reduces its complement-inhibiting effect [14, 15]. Bovine apo-lactoferrin, but not iron-saturated bovine lactoferrin, inhibited the classical but not the alternative pathway in standard hemolytic tests [16]. The cited study demonstrated a less pronounced effect of N-terminal peptides of bovine and human lactoferrins — lactoferricins B and H, respectively. Lactoferricin B, but not lactoferricin H, inhibited the killing of Escherichia coli by serum; the effect of non-fragmented lactoferrin was not described.

The interaction between lysozyme and complement involves the influence of lysozyme on complement activity, as well as synergism between lysozyme and MAC. First, hLZ has been characterized as a complement inhibitor, since high concentrations of the protein (500–1500 μg/mL) suppressed the hemolytic activity of blood serum in a classical pathway model using antibody-sensitized sheep erythrocytes [17]. Second, a group of studies focused on the combined effect of complement and lysozyme against Gram-negative bacteria. It is not obvious that bacteriolysis and killing of bacteria can be regarded as the same process, since killing of bacteria may not be accompanied by destruction of the cell membrane; in principle, destabilization of the components of the cell surface apparatus and loss of viability are different phenomena. Early studies demonstrated the synergism of serum and hLZ and HEWL in bacteriolysis [18–20]. A recent study has confirmed the synergism between MAC and hLZ at a higher methodological level [21]. The mechanism of synergy involves the incorporation of MAC into the outer membrane, which makes the peptidoglycan layer accessible to lysis by lysozyme, after which MAC and/or other lytic components of serum can destroy the inner membrane.

The existence of synergy between complement and lysozymes in killing bacteria remains less clear. Inoue et al. (1959) showed that lysozyme is not required for the bactericidal effect of human serum, and only an excess of the enzyme in diluted serum can enhance killing [18]. Unfortunately, a non-specific sorbent, bentonite, was used to deplete the sera of lysozyme, which can also remove other cationic antimicrobial polypeptides. In another study, the addition of high concentrations of HEWL to diluted serum at physiological ionic strength did not alter the killing kinetics of E. coli [22]. Martinez and Carrol (1980) showed that inhibition of hLZ by specific purified IgG did not affect the bactericidal activity of serum [20]. The authors concluded that lysozyme and complement exhibit synergy in bacteriolysis but not in killing in serum. In a recent study, specific removal of lysozyme from serum did not alter its bactericidal activity against E. coli and Stenotrophomonas maltophilia; however, no study of killing kinetics was conducted [23]. In a study by Glynn and Milne (1967), human serum depleted of lysozyme was obtained using bentonite or rabbit antiserum against hLZ (rabbit lysozyme was removed with bentonite) [19]. The authors showed that adding HEWL to hLZ-depleted serum restored its bactericidal activity. Similar results were obtained in another study [24]. Schiller et al. (1984) showed that bentonite treatment moderately reduced the bactericidal activity of diluted serum against Pseudomonas aeruginosa, but the kinetics of killing were not investigated [25].

A major drawback of certain cited studies is that hLZ and HEWL are considered interchangeable proteins, which may be incorrect, as they may affect the antimicrobial components of serum differently. Furthermore, the experiments were conducted at low ionic strength and/or in diluted serum; in certain cases, the absence of lysozyme was achieved by treating the serum with bentonite, which is a non-selective sorbent.

So, existing data show that the interaction between lysozyme and complement at different stages is conflicting: inhibition of cascade activation, but synergy with MAC in membrane lysis. However, the synergism between complement and hLZ or HEWL in serum under conditions close to physiological ones in killing bacteria remains questionable.

It cannot go unnoticed that in the literature, the activities of lactoferrin and lysozyme are considered separately, while in natural sources (phagocytes and gland secretions) these two proteins are co-localized. Moreover, a lactoferrin and lysozyme complex is known [26, 27]. The combined effect of the two proteins on complement has not been studied and needs to be clarified.

The aim of this study was to characterize the functional interaction of lactoferrin and lysozymes with the complement.

Materials and methods

Lactoferrin was isolated from breast milk using ion exchange chromatography and gel filtration, as described previously [28]. Lactoferrin saturation with iron, estimated by the OD₂₈₀/OD₄₆₆ ratio [29], was ~15%. Lysozyme was isolated from human oral fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography, as described previously [30]. A commercial preparation of HEWL (Sisco Research Laboratories, India) was used as a reference protein. The Escherichia coli ML-35p bacterial strain was kindly provided by Prof. R. Lehrer from the University of California (Los Angeles, USA).

The modulation of complement activity by lactoferrin and lysozymes was studied as described previously [31]. Pooled human blood serum served as a source of complement, and erythrocytes were used as a platform for complement activation and as a target for its lytic effect. In the classical pathway model, the proteins under study were added to serum (final concentration 1%) and antibody-sensitized sheep erythrocytes in 5 mM veronal buffer containing 15 mM dextrose, 0.05% gelatin, 0.15 M NaCl, 0.15 mM CaCl₂, 1 mM MgCl₂, pH 7.4. In the alternative pathway model, proteins were added to serum (final concentration 5%) and rabbit erythrocytes in 5 mM Veronal buffer containing 0.05% gelatin, 0.15 M NaCl, 10 mM MgCl₂, 10 mM EGTA, pH 7.4. The samples were incubated for 30 min at 37°C, and the reaction was stopped with PBS containing 30 mM EDTA. Complement activity was determined by serum hemolytic activity and the production of anaphylatoxins C3a and C5a. The latter was determined using a sandwich-type ELISA [32]. Briefly, aliquots of samples were added to wells of a plate coated with monoclonal antibodies to C3a or C5a after hemolytic tests, incubated for 1 hour at 37°C, washed with wash buffer to remove unbound antigens, and a conjugate of monoclonal antibodies with horseradish peroxidase was added. After one hour of incubation, the wells were washed to remove unbound conjugate, and a substrate mixture containing 3,3',5,5'-tetramethylbenzidine and H₂O₂ was added. The reaction was stopped with a stop reagent.

Samples without added proteins were used as controls in complement modulation experiments. If an increase in complement activity was observed, all effects were also evaluated in serum treated with 30 mM EDTA, a chelator of divalent ions that inactivates complement. Additionally, the possible intrinsic hemolytic activity of proteins was also controlled in serum heated at 57°C for 30 min to inactivate the complement. Complement activity was expressed as a percentage of the control and presented as the arithmetic mean ± standard deviation.

The bactericidal activity of serum, lactoferrin, and lysozymes was evaluated in experiments using round-bottomed wells with colony seeding and counting [33]. A 5 mM veronal buffer containing 0.15 M NaCl, 0.15 mM CaCl₂, 1 mM MgCl₂, pH 7.4 (VBS⁺⁺) was used in the experiments. E. coli ML-35p (10⁶ CFU/mL) in the logarithmic growth phase was incubated in buffer (control) or with the proteins under study at a concentration of 160 μg/mL (alone or in combination) with or without the addition of 5% or 10% serum. In certain samples, preheated 5% serum was used to evaluate the antimicrobial activity of lysozyme under inactive complement conditions. The total volume of each sample was 20 μL. The samples were incubated for 1 hour at 37°C, diluted with VBS⁺⁺, 10 μL was inoculated onto 40 mm diameter Petri dishes and poured with Trypticase Soy Agar Medium (Himedia, India). After overnight incubation, the colonies were counted. Bacterial survival was expressed as a percentage of the control as an arithmetic mean ± standard deviation.

To evaluate the killing kinetics, E. coli ML-35p was incubated in 50% serum containing or not containing 160 μg/mL of hLZ or HEWL. A series of identical samples were prepared, and aliquots were taken every 10 minutes for 1 hour. The aliquots were diluted with buffer and inoculated onto Petri dishes for further colony counting. The remaining details of the experiment (buffer composition, sample volume, incubation temperature, culture medium, sample dilution during seeding) were as in the previous paragraph.

The E. coli ML-35p strain is suitable for studying membrane permeabilization because it is deficient in lactose permease and contains periplasmic β-lactamase [34]. The bacteria were cultured in a medium containing ampicillin. To assess the permeability of the outer membrane, the β-lactamase substrate CENTA (Aurora Medbiochem Co., San Diego, USA) was used, and for the inner membrane, the β-galactosidase substrate resorufin-β-D-galactopyranoside (R-Gal; Abcam, USA) was used. These substrates were used because they are practically unmodified by serum. The products of their hydrolysis were detected at wavelengths of 407 or 573 nm, respectively. In a total volume of 200 μL, the final concentrations of the components were as follows: 75 μg/mL CENTA, 6 μg/mL R-Gal, 5% native or preheated serum, 5 ∙ 10⁵ CFU/mL E. coli, 160 μg/mL of the proteins under study, 0.15 M NaCl. The experiments used 5 mM veronal buffer (pH 7.4). Certain samples contained buffer instead of serum to assess membrane permeability in the absence of serum lytic factors. Samples in a flat-bottomed well plate were incubated at 37°C for 4 hours in a POLARstar Omega spectrophotometer (BMG LABTECH). The rate of membrane destruction was characterized by the t_0.5 value, which is the time required to reach ~50% of the maximum optical density. Optical density values between 10% and 90% of the maximum were used to calculate the z value:

$z=\frac{OD}{O{D}_{\max }-OD}.$

Then, the log10 z values were plotted on a graph as a function of log10 time (min) to linearize the data and determine the point of intersection with the x-axis, i.e., the point corresponding to the time of half-permeabilization (t_0.5). It is important to note that the graphs obtained in the described experiments do not reflect the true complement activity (MAC incorporation into membranes) or enzymatic kinetics. They reflect a complex process involving the effect of MAC, other lytic factors, and the activity of bacterial enzymes.

The muramidase activity of hLZ and HEWL was compared directly in a turbidimetric test. 10 μL of lysozymes (160 μg/mL) were added to 2 mL of Micrococcus lysodeikticus suspension (0.5 mg/mL) in VBS⁺⁺. The turbidity in the samples was measured as light scattering at a wavelength of 540 nm every 5 min for 20 min. Given the similar molecular weights of hLZ and HEWL, the final mass concentrations yielded equal molar concentrations.

Data processing and visualization were performed using the R language (v4.3.0) in the integrated development environment RStudio (v2023.03.0) using the ggplot2 (v3.5.2), ggpubr (v0.6.0), and ggpattern (v1.1.4) packages.

Results

Modulation of classical and alternative complement pathway activity by innate immunity proteins

In standard hemolytic tests, lactoferrin behaved as an inhibitor of the classical pathway. The IC₅₀ value was ~160 μg/mL (~2 μM) at the lysis level and ~20 μg/mL (~0.25 μM) at the anaphylatoxin production level. Lactoferrin did not affect the activity of the alternative pathway (Fig. 1, a–c). Human lysozyme at high concentrations (40–160 μg/mL, corresponding to ~2.7–11 μM) moderately enhanced the activation of both complement pathways, increasing C3a production (40–160 μg/mL or 160 μg/mL in models of the classical and alternative pathways) and C5a (160 μg/mL), as well as serum hemolytic activity (80–160 μg/mL) by 1.5–2 times compared to the control (Fig. 1, d–f). Importantly, all effects were abolished in EDTA-treated serum; and there were no hemolytic effects in preheated serum either. Consequently, lysozyme does not increase the signal in the ELISA system and is not hemolytic per se; instead, its enhancing effect is related to its effect on the complement. At the same time, HEWL did not affect complement activity parameters in two models (Fig. 1, g–i).

 

Fig. 1. Modulation of the activity of classical and alternative pathways by innate immunity proteins.

Modulation of complement activity by lactoferrin (a–c), hLZ (d–f), and HEWL (g–i). Complement activity was assessed by the production of anaphylatoxins C3a (a, d, g) and C5a (b, e, h), as well as by serum hemolytic activity (c, f, i). The significance of differences from samples without protein addition was assessed using a two-sample Student's t-test (* — p < 0.05).

 

Joint modulation of classical complement pathway activity by lactoferrin and hLZ

As a logical extension of the study, we conducted a single experiment to investigate the combined effect of lactoferrin and hLZ on the classical pathway. The results are shown in Fig. 2. Lactoferrin at a concentration of 4 μM (320 μg/mL) reduced hemolysis to ~20% of the control level. 4 μM lysozyme had no detectable effect, but 8 μM lysozyme slightly increased the hemolytic activity of serum. These effects are consistent with the results shown in Fig. 1. The simultaneous presence of two proteins in equimolar concentrations (4 μM) resulted in an effect comparable to the presence of lactoferrin alone. The addition of a twofold molar excess of lysozyme to 4 μM lactoferrin increased complement activity, which was still inhibited by lactoferrin. The increase in hemolytic activity was approximately equal to the potentiating effect of 8 μM lysozyme. Therefore, even in molar excess, lysozyme did not abolish the inhibition of complement by lactoferrin. Based on a single experiment, it can be concluded that the two proteins modulate complement independently.

 

Fig. 2. Hemolytic activity of serum in the classical pathway model in the presence of lactoferrin and human lysozyme. Results of a single experiment. Abbreviations: LF — lactoferrin, hLZ — human lysozyme.

 

Bactericidal activity of serum and innate immunity proteins against E. coli ML-35p

We then characterized the bactericidal activity of three proteins and human blood serum against E. coli. The results are shown in Fig. 3. Lactoferrin and HEWL in buffer had a weak feeding effect, stimulating bacterial growth. 5% native serum, including in the presence of lactoferrin, slightly increased bacterial growth. However, the addition of hLZ or HEWL significantly reduced bacterial survival, and the effects of the two proteins were comparable. HEWL did not contribute to the killing of bacteria in preheated serum, which was also nourishing. Lactoferrin partially and significantly counteracted the synergistic effect of lysozymes and 5% native serum. 10% serum effectively killed bacteria. Lactoferrin did not cancel its bactericidal effect. Since 10% serum was quite effective on its own, it is difficult to assess the effect of lysozymes under these conditions. This necessitates characterizing the effect of lysozymes in a serum concentration close to physiological and studying the kinetics of the bactericidal effect.

 

Fig. 3. Bactericidal activity of serum, lactoferrin, and lysozymes against E. coli ML-35p.

Bacteria (10⁶ CFU/mL) were incubated in VBS⁺⁺ or in 5% or 10% serum in the presence or absence of proteins of interest for 1 hour at 37 °C; control — bacteria in VBS⁺⁺ in the absence of proteins. The final concentrations of the proteins under study were 160 μg/mL. After incubation, the suspensions from the wells were diluted and inoculated onto Petri dishes. Colonies were counted after overnight incubation. A two-sample Student's t-test was performed for selected groups; p-values are shown in the graph. Abbreviations: LF — lactoferrin, HEWL — hen egg white lysozyme, hLZ — human lysozyme.

 

Study of the permeability of E. coli ML-35p membranes

We also characterized the effect of lysozyme and lactoferrin, individually or in combination, in buffer or serum on the permeability of E. coli membranes. Lactoferrin and HEWL did not increase the permeability of the outer and inner membranes in either buffer or preheated serum (Fig. 4, a, b, e, f). Native serum increased the permeability of the outer and inner membranes (Fig. 4, c, d). Lactoferrin and HEWL did not alter the rate of permeabilization of the outer membrane by native serum, but HEWL accelerated the destruction of the inner membrane regardless of the presence of lactoferrin (Fig. 4, c, d, g–i). After the experiments, the bacterial colonies were counted. Native serum reduced bacterial survival; while HEWL further moderately reduced survival, lactoferrin tended to increase bacterial survival, and both proteins increased survival in preheated serum (Fig. 4, j).

 

Fig. 4. Results of bacterial membrane permeabilization analysis (experiments with HEWL).

E. coli ML-35p (5 ∙ 10⁵ CFU/mL) was incubated in buffer (a, b), native 5% serum (c, d) or preheated serum (e, f) in the absence or presence of 160 μg/mL hen egg white lysozyme (HEWL), lactoferrin (LF), or a combination thereof. The permeability of the outer membrane (a, c, e) or inner membrane (b, d, f) was determined by assessing CENTA or R-Gal hydrolysis, respectively. The data obtained from the native serum samples were linearized to calculate t_0.5, which characterizes the rate of permeabilization of the outer (g) or inner membrane (h). The calculated t_0.5 values are presented on a heat map; the control is the effect of serum without proteins of interest (i). After incubation, the suspensions from the wells with CENTA or R-Gal were diluted and the colonies were counted after overnight incubation in agarose medium (j). Survival results were obtained in two independent experiments conducted in two parallels (i.e., in the presence of CENTA or R-Gal).

 

Human lysozyme did not permeabilize the membranes per se; it accelerated the destruction of the inner, but not the outer, membrane by native serum (Fig. 5).

 

Fig. 5. Results of bacterial membrane permeabilization analysis (experiments with hLZ).

E. coli ML-35p (5 × 10⁵ CFU/mL) was incubated in buffer (a, b), native 5% serum (c, d) or preheated serum (e, f) in the absence or presence of 160 μg/mL lysozyme. The permeability of the outer membrane (a, c, e) or inner membrane (b, d, f) was determined by assessing CENTA or R-Gal hydrolysis, respectively. The data obtained from samples in native serum were linearized to calculate t_0.5, which characterizes the rate of permeabilization of the outer (g) or inner membrane (h). The calculated t_0.5 values are presented on a heat map; control — the effect of serum without proteins of interest (i).

 

Kinetics of bacterial killing by serum

Based on the data obtained, it was hypothesized that the biological effect of lysozymes could lead to accelerated killing of bacteria. To clarify the effect of hLZ and HEWL on the kinetics of bacterial killing, taking into account the presence of nutritious and bactericidal components of serum, we used 50% serum, since its concentration is close to the physiological concentration of blood plasma. We found that this proportion of serum killed almost all bacteria within 40 minutes. By the 20th minute, the serum had no effect on bacterial survival, while the presence of HEWL led to the destruction of more than 50% of the bacteria. Moreover, hLZ (capable of enhancing the effect of the complement) accelerated the killing of bacteria better than HEWL; three-quarters of the bacteria were killed within 10 minutes (Fig. 6). The kinetic curves in Fig. 6 clearly demonstrate the effect of muramidases on the killing of bacteria by serum, as well as the differences between enzymes that have the above-described different effects on complement.

 

Fig. 6. Kinetics of serum bactericidal activity. E. coli ML-35p (10⁶ CFU/mL) was incubated in 50% serum in the absence or presence of 160 μg/mL of hLZ or HEWL for specified time intervals, diluted, inoculated onto Petri dishes and colonies were counted after overnight incubation.

 

The difference between the effects of hLZ and HEWL on the kinetics of bacterial killing by serum could be explained by differences in enzymatic activity. We directly compared the muramidase activity of the two proteins in a turbidimetric test in VBS⁺⁺, i.e., in the buffer used in all antimicrobial tests. No differences in the enzymatic activity of the two proteins were found. (Fig. 7).

 

Fig. 7. Muramidase activity of HEWL and hLZ.

The two proteins at equal mass/molar concentrations were mixed with Micrococcus lysodeikticus suspension, and turbidity at 540 nm was measured at specified time intervals.

 

Discussion

In this study, the combined effect of lactoferrin and lysozymes in the context of interaction with the complement system was investigated.

We confirmed data showing that lactoferrin (a natural mixture with a predominant apo-form) is a selective inhibitor of the classical pathway (Fig. 1, a–c). We demonstrated the ability of lactoferrin not only to inhibit MAC assembly but also to suppress the production of anaphylatoxins. This is consistent with the literature data on the effect of lactoferrin at the level of C3 convertase of the classical pathway [14, 15]. This function of apo-lactoferrin can be called anti-inflammatory. It is consistent with the ability of apo-lactoferrin to prevent the formation of hydroxyl radicals in the Haber–Weiss reaction by binding iron ions [35].

Although lactoferricin B has been shown to inhibit complement-mediated killing of E. coli, lactoferricin H has been found to lack this ability [16]; the cited study did not report on the effect of intact lactoferrin on serum-mediated bacterial killing. Therefore, we sought to determine whether lactoferrin could inhibit the killing of bacteria by serum complement. Bacteria were nourished in 5% serum, while their viability was significantly reduced in 10% serum. Lactoferrin in buffer and in 5% serum stimulated bacterial growth, presumably as a simple nutrient, since bovine serum albumin at high concentrations behaved similarly (data not shown). Lactoferrin did not inhibit the killing of bacteria in the presence of 10% serum. Therefore, inhibition of the classical complement pathway by lactoferrin is not significant in the context of bacterial killing. The most likely explanation is that the alternative pathway, rather than the classical pathway, is primarily responsible for complement activation on the surface of bacteria, while lactoferrin inhibits only the classical pathway.

The study also revisited the function of lysozyme as a complement modulator. An earlier study demonstrated the inhibition of the classical pathway by lysozyme [17]. Unexpectedly, it was shown that hLZ, but not HEWL, activates both the classical and alternative complement pathways. The contradiction between the literature data and our results may be due to differences in the concentration ranges studied, i.e., 50–1500 μg/mL in the cited study and 1.25–160 μg/mL in our study. Furthermore, the exact concentration of blood serum was not specified in the cited study, but ~50% lysis was achieved after 60 min of incubation; therefore, most likely, the serum concentration was less than 1%, which further increased the lysozyme : complement ratio, predisposing the system to nonspecific effects. According to our results, hLZ enhanced the production of both anaphylatoxins, and this occurred in two models, so we suggest that lysozyme acts at the level of C3 conversion. Considering the homology between C3 and C4, as well as between factor B and C2, the binding of lysozyme to several targets involved in the classical and alternative pathways may explain the effect of lysozyme on complement activity. Although we have not identified specific targets of lysozyme in blood serum, it is logical to assume that differences between the complement-modulating abilities of hLZ and HEWL may be due to differences in their structures. The alignment of the primary structures and comparison of the tertiary structures of the two lysozymes are presented in Fig. 8. The question remains open as to whether complement modulation by lysozymes is evolutionarily conservative; for example, whether HEWL could influence complement activation in chickens.

 

Fig. 8. Comparison of hLZ and HEWL structures.

The panel above: sequences alignment of hLZ (higher sequence, UniProt P61626) and HEWL (lower sequence, UniProt P00698). Numeration of amino acid residues is given for the longer sequence, i.e. hLZ; HEWL lacks residue 48 compared to human homolog. Black lines denote disulfide bonds. Below: comparison of tertiary structures of hLZ (left, Protein Data Bank 2BQA) and HEWL (right, Protein Data Bank 1DPX). N- and C-termini are denoted. Differing amino acid residues with similar physicochemical propertied are colored in magenta; residues with dissimilar properties are colored in yellow. Visualization was performed with Visual Molecular Dynamics v1.9.3.

 

Both hLZ and HEWL killed bacteria in the presence of 5% serum. The effect was observed only in native, but not preheated serum, indicating interaction with complement.

The results of this study confirm the model in which lysozymes interact with MAC to accelerate the permeability of the inner membrane of Gram-negative bacteria. We demonstrated this effect using both HEWL and hLZ. Although lysozymes themselves do not destroy the outer membrane, MAC is capable of doing so. It provides access for lysozymes to the periplasmic space. The faster destruction of the inner membrane can be explained by the lysis of the peptidoglycan layer by muramidases, which facilitates the incorporation of MAC and/or other lytic agents into the inner membrane. HEWL and hLZ accelerated the permeabilization of the inner membrane by serum at a comparable rate, but hLZ appeared to have a slightly more pronounced effect than HEWL. No difference was found in the rate of outer membrane destruction by serum in the absence and presence of hLZ, despite its ability to enhance complement activity. This may be due to methodological shortcomings associated with the determination of the reaction product catalyzed by β-lactamase.

It was hypothesized that lactoferrin's ability to increase bacterial viability in the presence of serum and lysozyme (Fig. 3) is associated with the formation of a large complex that hinders passage through the MAC pore or prevents the interaction of muramidases with the substrate. However, lactoferrin did not prevent the accelerated destruction of internal membranes by serum and HEWL, which leads to the conclusion that its ability to increase bacterial survival is due to its own nourishing effect.

Both lysozymes accelerated the killing of bacteria by 50% with serum, but with varying degrees of effectiveness: hLZ did this better than HEWL. At the same time, the muramidase activity of the two lysozymes was the same under the experimental conditions. Therefore, the differences in killing acceleration cannot be explained by differences in muramidase activity; instead, we believe that they depend on the ability to modulate complement.

Taking into account the studies mentioned in the Introduction of this article [18–25], to our knowledge, this is the first study demonstrating the synergistic bactericidal effect of complement and lysozymes in 50% serum at physiological ionic strength, and the first study comparing hLZ and HEWL under these conditions. We demonstrated synergism both in the assessment of survival after incubation and in the experiment to study the kinetics of bacterial killing. It is important to note that lysozyme-depleted serum was not used; instead, exogenous lysozyme was added to normal serum to mimic neutrophil degranulation and functional interaction of the protein with plasma components diffusing into the site of inflammation.

Increased production of anaphylatoxins in the presence of lysozyme may contribute to the recruitment and activation of innate immune cells; C4b, C3b, and other surface-bound derivatives may participate in opsonophagocytosis. Moreover, human C4a and C3a exert a direct antimicrobial effect through membrane lysis [36–39]. We suggest that these mechanisms, triggered in the presence of lysozyme, may additionally contribute to the elimination of pathogens, although they have not been studied in the present study.

Based on our results, we propose a new model in which lysozymes cooperate with complement to lyse and kill bacteria through three mechanisms:

• synergism with MAC;
• enhancement of the classical pathway;
• enhancement of the alternative pathway.

From two perspectives, namely complement modulation and antimicrobial activity, the behavior of human lactoferrin and lysozyme can be described as partial antagonism: lactoferrin is an inhibitor of the classical pathway (anti-complement factor), while lysozyme is an activator of the classical and alternative pathways (pro-complement factor); lysozyme interacts with complement to accelerate bacterial killing, while lactoferrin can serve as a nutrient for certain bacteria, at least in vitro. However, their antagonism is limited by two facts. First, lactoferrin and lysozyme modulate the classical complement pathway independently: they do not enhance or weaken the effect of one another. Second, although lactoferrin promotes the growth of E. coli in diluted serum due to its feeding effect, it does not prevent the acceleration of internal membrane destruction due to the cooperation of lysozyme with complement. Thus, we can assume that the resulting effect of the two proteins in vivo depends on the ratio of their concentrations. It is important to note that lactoferrin has antimicrobial activity against certain types and strains of microbes and can act synergistically with lysozyme, as has been shown in experiments against pneumococci [40]. In this regard, our results should be extrapolated to other microbes with great caution.

According to the literature sources, lactoferrin acts as a complement inhibitor at the C3 conversion level [14, 15]. We also assumed that hLZ acts at the same level (see above). However, these two proteins do not interfere with each other's effects on the classical pathway. Considering also the possibility of lactoferrin-lysozyme complex formation [26, 27], this makes the identification of lactoferrin and lysozyme targets among complement proteins an intriguing and promising task.

The results obtained highlight the importance of experimental factors such as (a) the balance of nutritional and bactericidal factors depending on the concentration of serum and additional proteins, and (b) incubation time. More specifically, 5% serum increased bacterial viability after one hour of incubation, but partially reduced it after four hours of incubation. Furthermore, after one hour of incubation, 5% serum enhanced bacterial growth, while 10% serum killed them; the 50% serum killed almost all bacteria within 40 minutes. The higher the serum concentration and the longer the bacteria remain in it, the more pronounced the predominance of bactericidal activity over the nourishing activity of serum in vitro. However, the nutritional effect of preheated serum after 4 hours of incubation was no more pronounced than after 1 hour of incubation with bacteria.

Conclusion

This study partially confirms, partially refutes and expands on previously described biological activities of lactoferrin and lysozymes in the context of complement. In particular, we clarify the understanding of the functional interaction between complement and hLZ. According to the proposed model, lysozyme cooperates with complement not only at the level of the membrane attack complex during bacteriolysis, but also at earlier stages of complement activation, as confirmed by the enhanced conversion of C3 and C5 proteins upon the classical and alternative pathways activation. Lysozyme contributes to the acceleration of bacterial killing in the presence of complement. There is also emphasis on the importance of species differences in homologous proteins using the example of hLZ and HEWL, as well as in vitro experimental conditions for interpreting data on the functional effects of these innate immunity proteins.

About the authors

Ilia A. Krenev

Institute of Experimental Medicine

Author for correspondence.
Email: il.krenevv13@yandex.ru
ORCID iD: 0000-0001-7970-3291

junior researcher, Laboratory of general pathology

Russian Federation, Saint Petersburg

Ekaterina S. Umnyakova

Institute of Experimental Medicine

Email: ekaterina.umnyakova@unibas.ch
ORCID iD: 0000-0002-0311-3305

Cand. Sci. (Biol.), senior researcher, Laboratory of general pathology

Russian Federation, Saint Petersburg

Alexey V. Sokolov

Institute of Experimental Medicine; Saint Petersburg State University

Email: biochemsokolov@gmail.com
ORCID iD: 0000-0001-9033-0537

Dr. Sci. (Biol.), Professor, Head, Laboratory of analysis of intermolecular interactions, Institute of Experimental Medicine; Professor, Department of biochemistry, Faculty of biology, Saint Petersburg State University

Russian Federation, Saint Petersburg; Saint Petersburg

Nikolay P. Gorbunov

Saint Petersburg Pasteur Institute

Email: niko_laygo@mail.ru
ORCID iD: 0000-0003-4636-0565

junior researcher, Laboratory of hybridoma technologies

Russian Federation, Saint Petersburg

Valeria A. Kostevich

Saint Petersburg Pasteur Institute

Email: hfa-2005@yandex.ru
ORCID iD: 0000-0002-1405-1322

Cand. Sci. (Biol.), senior researcher, Laboratory of hybridoma technologies

Russian Federation, Saint Petersburg

Galina M. Aleshina

Institute of Experimental Medicine

Email: galina_aleshina@mail.ru
ORCID iD: 0000-0003-2886-7389

Dr. Sci. (Biol.), Associate Professor, Head, Laboratory of general pathology

Russian Federation, Saint Petersburg

Mikhail N. Berlov

Institute of Experimental Medicine; Saint Petersburg State University

Email: berlov.mn@iemspb.ru
ORCID iD: 0000-0001-5191-0467

Cand. Sci. (Biol.), leading researcher, Laboratory of general pathology

Russian Federation, Saint Petersburg; Saint Petersburg

References

Supplementary files