Porcine and Bovine Forms of Lactoferrin Inhibit Growth of Porcine Enterotoxigenic Escherichia coli and Degrade Its Virulence Factors

Currently, postweaning F4⁺ and F18⁺ Escherichia coli infections in piglets are controlled by the use of antibiotics and zinc oxide, but the use of these antimicrobial agents most likely contributes to an increase in antibiotic resistance. Our work demonstrates that bovine and porcine lactoferrin can inhibit the growth of porcine enterotoxigenic E. coli strains. In addition, we also show that lactoferrin can reduce the adherence of these strains to small intestinal epithelial cells, even at a concentration that does not inhibit bacterial growth. This research could allow us to develop lactoferrin as an alternative strategy to prevent enterotoxigenic E. coli (ETEC) infections in piglets.

ABSTRACT

Postweaning diarrhea (PWD) is an economically important, multifactorial disease affecting pigs within the first 2 weeks after weaning. The most common agent associated with PWD is enterotoxigenic Escherichia coli (ETEC). Currently, antibiotics are used to control PWD, and this has most likely contributed to an increased prevalence of antibiotic-resistant strains. This puts pressure on veterinarians and farmers to decrease or even abandon the use of antibiotics, but these measures need to be supported by alternative strategies for controlling these infections. Naturally derived molecules, such as lactoferrin, could be potential candidates due to their antibacterial or immune-modulating activities. Here, we analyzed the ability of bovine lactoferrin (bLF), porcine lactoferrin (pLF), and ovotransferrin (ovoTF) to inhibit ETEC growth, degrade ETEC virulence factors, and inhibit adherence of these pathogens to porcine intestinal epithelial cells. Our results revealed that bLF and pLF, but not ovoTF, inhibit the growth of ETEC. Furthermore, bLF and pLF can degrade several virulence factors produced by ETEC strains, more specifically F4 fimbriae, F18 fimbriae, and flagellin. On the other hand, ovoTF degrades F18 fimbriae and flagellin but not F4 fimbriae. An in vitro adhesion assay showed that bLF, ovoTF, and pLF can decrease the number of bacteria adherent to epithelial cells. Our findings demonstrate that lactoferrin can directly affect porcine ETEC strains, which could allow lactoferrin to serve as an alternative to antimicrobials for the prevention of ETEC infections in piglets.

IMPORTANCE Currently, postweaning F4⁺ and F18⁺ Escherichia coli infections in piglets are controlled by the use of antibiotics and zinc oxide, but the use of these antimicrobial agents most likely contributes to an increase in antibiotic resistance. Our work demonstrates that bovine and porcine lactoferrin can inhibit the growth of porcine enterotoxigenic E. coli strains. In addition, we also show that lactoferrin can reduce the adherence of these strains to small intestinal epithelial cells, even at a concentration that does not inhibit bacterial growth. This research could allow us to develop lactoferrin as an alternative strategy to prevent enterotoxigenic E. coli (ETEC) infections in piglets.

INTRODUCTION

Postweaning diarrhea (PWD) is a multifactorial disease and one of the major causes of economic loss for the swine industry, affecting piglets in the first 2 weeks after weaning. This disease is characterized by a watery diarrhea leading to dehydration, growth retardation, and even mortality in piglets (1). The most common agents associated with this disease are enterotoxigenic Escherichia coli (ETEC) bacteria (1). These ETEC strains produce heat-labile (LT) and/or heat-stable enterotoxins (STa and STb) and colonization factors or fimbriae. Both the enterotoxins and fimbriae are involved in the development of the disease (2). These fimbriae are long filamentous protein polymers and are essential to the ability of ETEC to adhere to small intestinal enterocytes. Porcine ETEC strains mainly express two types of fimbriae, F4 and F18 fimbriae (3). F4 fimbriae are composed of the major structural subunit FaeG, which also functions as the adhesin, and several minor subunits (4). F18 fimbriae, on the other hand, are composed of the major structural subunit FedA and the minor subunits FedE and FedF. FedF functions as the tip adhesin of F18 fimbriae (5). Both F4 and F18 fimbriae bind to specific glycosylated receptors present in the apical membrane of small intestinal epithelial cells (4, 6). The presence of these fimbrial receptors is essential in determining the susceptibility of piglets to F4⁺ and F18⁺ ETEC infections (2). Furthermore, ETEC strains can also produce flagella, which consist of several structural and regulatory proteins, including flagellin. These flagella, in addition to their known motility function, can also participate in biofilm formation and adhesion (7).

Extensive use of antibiotics and zinc oxide during the first 2 weeks after weaning is common to control PWD and has most likely contributed to an increased occurrence of multiple antibiotic-resistant bacterial strains (8–11). The emergence of multiple-antibiotic-resistant strains and the appearance of colistin resistance genes on transferable plasmids of E. coli from the gut of pigs is especially concerning, as colistin is widely used in animals but is also a last-resort antibiotic to treat infections of humans with multidrug-resistant bacteria (9, 12). In this context, antimicrobial resistance (AMR) is recognized as a global threat, and efforts in combatting AMR were implemented, including the 2015 WHO Global Action Plan on AMR and the 2016 United Nations Political Declaration (13, 14). Boosting research on the development of alternatives to antibiotics is a major action point in all these programs. Naturally derived molecules that can decrease infection by a direct effect on the microorganisms and/or increase host resistance have recently gained interest. One of those molecules is lactoferrin.

Lactoferrin (LF), an iron-binding glycoprotein, is present in exocrine secretions of mammals, such as milk. Currently, LF is found in nine species, including human, cow, pig, horse, goat, and mouse. The amino acid sequences are highly conserved, as a homology of between 65 and 90% is observed (15). Ovotransferrin (ovoTF), which belongs to the same family of transferrin iron-binding glycoproteins, is present in egg white and chicken serum (16). These glycoproteins play a role in iron homeostasis (17), cellular growth and differentiation (18), protection against viral and bacterial infections (19, 20), and anti-inflammatory activities (21, 22). Furthermore, lactoferrin has a bacteriostatic function due to the chelation of Fe³⁺ ions and a bactericidal activity through its binding to lipopolysaccharides (LPS) in Gram-negative bacteria and lipoteichoic and teichoic acids in Gram-positive bacteria (23). Both the full-length protein and lactoferrin-derived peptides, such as lactoferricin, disrupt the bacterial cell wall, resulting in an increase in membrane permeability (23). Furthermore, lactoferrin displays a proteolytic activity toward bacterial virulence factors (24–27). Bovine lactoferrin (bLF), for instance, degrades the type III secretion system molecules EspA and EspB of enterohemorrhagic E. coli (EHEC), which inhibited the attachment of these bacteria to intestinal epithelial cells. Both bLF and human lactoferrin (hLF) degraded EspA and EspB, but bLF was more effective (24, 27). Besides E. coli, bLF and ovoTF also inhibited the attachment and cell entry of Chlamydia psittaci in chicken macrophages. Interestingly, ovoTF was more effective than bLF (28). Overall, these results indicate that a transferrin of one species is more effective against pathogens of that species. In contrast to hLF and bLF, less is known about the capacity of porcine lactoferrin (pLF) to prevent E. coli infections in piglets. Only a limited number of studies have been performed on the mode of action of lactoferrin in pigs or on porcine pathogens. Recently, LFP-20, an antimicrobial peptide in the N terminus of pLF, was shown to inhibit MAPK and NF-κB signaling pathways, preventing LPS-induced inflammation in the pig alveolar macrophage cell line 3D4/2 (29). Moreover, LFP-20 protected the intestinal barrier by preventing the disruption of tight junctions by LPS (30). Here, we aimed to assess the effect of ovoTF, bLF, and pLF on the growth of F4⁺ and F18⁺ enterotoxigenic E. coli (ETEC) strains, their attachment to intestinal epithelial cells, and degradation of ETEC-associated virulence factors.

RESULTS

Production and purification of recombinant porcine lactoferrin.

Recombinant pLF was produced using a workflow developed by our laboratory (31). High-pLF-producing CHO clones were generated by transfecting CHO cells with a pLF- and enhanced green fluorescent protein (eGFP)-encoding construct (Fig. 1A). The transfected CHO cells were then cultured for several days and single-cell sorted into 96-well plates based on their GFP expression. We then selected GFP-positive clones based on the following criteria: (i) fast growing and (ii) high and homogeneous GFP expression levels, as determined by live-cell microscopy. The secretion of pLF by these clones in the culture medium was then detected by Western blot analysis using anti-V5 antibodies (Fig. 1B). This revealed that all the selected clones produced and secreted recombinant pLF, although at different levels. The highest-producing clone was then grown in suspension, and recombinant pLF was purified via its His tag.

FIG 1.

Production of recombinant porcine lactoferrin. (A) Schematic representation of the porcine lactoferrin construct integrated into the pcDNA3.1+N-eGFP vector. (B) Production and secretion of recombinant pLF by transfected CHO cells (clones: 1F1-2H9) was detected using an anti-V5 antibody. GFP, green fluorescent protein; TEV, tobacco etch virus sequence; V5, V5 tag; His, His tag; Ab, antibody 1F1; 2H9, transfected CHO cells.

Iron saturation of bovine and porcine lactoferrin and ovotransferrin.

As the antibacterial activity of lactoferrin in part depends on its ability to chelate ferric iron, we also determined the iron binding capacity of bLF, pLF, and ovoTF. To be able to evaluate this iron binding capacity of the recombinant-produced porcine lactoferrin, native porcine lactoferrin was purified from milk samples. The iron saturation of bLF, ovoTF, and recombinant and native pLF was determined using a high-throughput assay as previously described (32). The results showed that all lactoferrins bind ferric ions to similar extents (Table 1). Moreover, these data suggest that the recombinant pLF is properly folded and has an iron binding capacity similar to that of its native counterpart.

TABLE 1.

Iron saturation levels of bovine and porcine lactoferrin and ovotransferrin

Lactoferrina	Before (saturation %)	After (saturation %)
bLF	15.3	75.3
ovoTF	9.2	73.2
Rec pLF	12.6	67.8
Nat pLF	8.7	71.3

^abLF, bovine lactoferrin; ovoTF, ovotransferrin; Rec pLF, recombinant porcine lactoferrin; Nat pLF, native porcine lactoferrin.

Lactoferrin inhibits the growth of porcine ETEC strains.

Lactoferrins possess a bacteriostatic and a bactericidal function. Several ETEC strains (Table 2) were incubated with different concentrations of bLF, ovoTF, and native and recombinant pLF in order to determine the effect of LF. As shown in Fig. 2, bLF and pLF (both native and recombinant), at concentrations of 10 and 5 mg/ml, inhibited the growth of the ETEC strains GIS26 (Fig. 2), IMM01 (Fig. S1 in the supplemental material), and F107/86 (Fig. 2), while the growth of the E. coli strains H56 (Fig. S1), G7 (Fig. 2), and 2134P (Fig. 2) was only inhibited using the highest concentration of 10 mg/ml. However, the growth of G7 (Fig. 2) was minimally, but significantly, affected by native and recombinant pLF (at 5 mg/ml) at 2, 3, and 6 to 8 h and at 2, 3, 7, and 8 h, respectively. Remarkably, ovoTF did not inhibit the growth of these bacteria (Fig. 2).

TABLE 2.

Porcine-specific E. coli strains used for the growth inhibition assay

Strain	Serotype	Fimbriaa	Toxins	Reference
GIS26	O149:K91	F4ac	LT, pSTa, STb	58
IMM01	O149:K91	F4ac	LT, STb	58
G7	O8:K87	F4ab	LT	59
H56	O8:K87	F4ad		60
F107/86	O139:K12	F18ab	Stx2e	61
2134P	O157	F18ac	pSTa, STb	61

^aLT, heat-labile enterotoxin; ST, heat-stable enterotoxin. F4ab, F4ac, and F4ad are antigenic F4 fimbria variants that differ in the primary FaeG sequence.

FIG 2.

Inhibitory effect of bLF, ovoTF, and native and recombinant pLF on the growth of different E. coli strains. (Top to bottom) Effect of lactoferrins on the bacterial growth of GIS26, G7, F107/86, and 2134P E. coli strains when incubated with bLF, ovoTF, or pLF at different concentrations (mg/ml; see legend at top left). The optical density (OD) was measured spectrophotometrically at 595 nm every hour for 8 hours. The results are represented as the mean OD ± the standard error of the mean (SEM) (n = 3 independent experiments). The data in the rectangles are significantly different from the control (P < 0.05, 2-way ANOVA).

Degradation of ETEC virulence factors by lactoferrin.

ETEC strains produce fimbriae which enable them to adhere to specific receptors present in the apical membrane of small intestinal enterocytes. This interaction allows the bacteria to colonize the small intestine and to secrete enterotoxins, which trigger diarrhea (33, 34). Porcine ETEC strains mainly produce F4 and F18 fimbriae. Lactoferrins have a protease activity, and since we previously showed that bLF can degrade the EHEC-secreted effector proteins EspA and EspB (24), we wanted to evaluate the degradation of ETEC-associated virulence factors (fimbriae, flagellin, and enterotoxins) by ovotransferrin and bovine and porcine lactoferrin.

First, we evaluated the ability of ovoTF, bLF, and porcine lactoferrin (native and recombinant) to degrade purified F4 fimbriae. The latter are composed of repeating subunits of the major structural subunit FaeG, which also functions as an adhesin. Figure 3A shows that in contrast to ovoTF, bLF and native and recombinant pLF significantly degrade F4 fimbriae. As ovoTF did not degrade purified F4 fimbriae, we next determined that the native structure of FaeG does not shield potential ovoTF cleavage sites. To this end, F4 fimbriae were heat denatured and subsequently incubated with bLF or ovoTF (Fig. 3B). As expected, bLF degraded the denatured F4 fimbriae. However, ovoTF did not degrade denatured F4 fimbriae. Three antigenic F4 fimbrial variants, F4ab, F4ac, and F4ad, have been identified. These variants were purified from different ETEC strains (Table 2) and subsequently used in the in vitro degradation assay. No significant differences were observed in the ability of bLF to degrade these different F4 fimbrial variants (Fig. 3C). OvoTF did not degrade these variants. To ascertain that this degradation results from the protease activity of lactoferrin, we incubated purified F4 fimbriae with bLF and pLF in the absence or presence of 0.5 mM phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor. As shown in Fig. 3D, PMSF inhibited bLF- and pLF-mediated F4 fimbria degradation, demonstrating that the LF-mediated degradation of F4 fimbriae is the result of its proteolytic activity.

FIG 3.

The effect of lactoferrins on F4 fimbria degradation. (A to D) The relative quantification of (A) native F4 degradation by bLF, ovoTF, and native and recombinant pLF with a representative Western blot (right), (B) denatured F4 degradation by bLF and ovoTF with a representative Western blot (right), (C) different antigenic F4 fimbria variants (left) with representative Western blot (right), (D) F4 fimbria degradation in the absence or presence of PMSF with a representative Western blot (right). Lactoferrins at 1 mg/ml were used to assess F4 fimbriae degradation (n = 4), while a sample lacking lactoferrin was used as a control (No LF). (E) F4 fimbria degradation by different concentrations of bLF, ovoTF, and native and recombinant pLF (n = 4) with a representative Western blot (right). The concentrations (in mg/ml) of the lactoferrins are presented on the x axis or above the corresponding bands. A black tooling line was added between the blots (nat and rec pLF) to indicate that these are two separate blots which are placed together. Data in the graphs are normalized to their respective controls (no LF) and presented as the mean ± SD. A Kruskal-Wallis test with a correction for multiple comparisons was performed by controlling the false discovery rate using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *, q < 0.05 compared to the control; LF, lactoferrin; bLF, bovine lactoferrin; OvoTF, ovotransferrin; nat pLF, native porcine lactoferrin; rec pLF, recombinant porcine lactoferrin; M, MagicMark protein standard; no LF, no lactoferrin was added.

Next, degradation of purified F4 fimbriae at lower concentrations (1, 0.5, 0.25, and 0.1 mg/ml) of the lactoferrins was assessed. As shown in Fig. 3E, the proteolytic effect of bLF was observed when the concentration was decreased to 0.5 mg/ml (Fig. 3E). Moreover, both native and recombinant pLF degraded FaeG at concentration of 1, 0.5, and 0.25 mg/ml. OvoTF, on the other hand, did not degrade purified F4 fimbriae (Fig. 3E).

In addition to F4 fimbriae, we also assessed the capacity of bLF, ovoTF, and pLF (native and recombinant) to degrade F18 fimbriae. The latter are composed of multiple subunits of the major structural subunit FedA and the tip adhesin FedF. As shown in Fig. 4, ovoTF, bLF, and native and recombinant pLF at a concentration of 1 mg/ml significantly degraded both the FedF (Fig. 4A) and FedA (Fig. 4B) subunits. No significant differences were observed between the different lactoferrins. Furthermore, degradation of the F18 fimbrial subunits was evaluated at lower concentrations of lactoferrins (1, 0.5, 0.25, 0.1, and 0.01 mg/ml). When the concentration of bLF, ovoTF, and porcine lactoferrin (native and recombinant) was decreased to 0.5 mg/ml, FedF degradation was not observed (Fig. 4C). In contrast, the FedA subunit was still degraded by bLF and ovoTF at 0.5 mg/ml and by native and recombinant pLF at 0.5 and 0.25 mg/ml (Fig. 4D). To assess that the observed F18 fimbria degradation results from the protease activity of lactoferrin, we incubated purified F18 fimbriae with the different lactoferrins in the absence or presence of PMSF. As shown in Fig. 4E, PMSF inhibited LF-mediated degradation, demonstrating that the observed F18 fimbria degradation is due to the proteolytic activity of LF.

FIG 4.

The effect of different lactoferrin concentrations on FedF and FedA degradation. The relative quantification of FedF (A and C) and FedA (B and D) degradation with, on the right, representative Western blots. (A, B, and E) Lactoferrins at 1 mg/ml were used to assess F18 fimbria degradation, while a sample lacking lactoferrin was used as a control (no LF). (C and D) The concentrations (in mg/ml) of the lactoferrins are presented on the x axis or above the corresponding bands. Data in the graphs are normalized to their respective controls (no LF) and presented as the mean ± SD based on 4 independent experiments. In panel B, no molecular ladder was present on the Western blot, so an overlay was created between the prestained ladder and the Western blot image in order to determine the molecular weight of the observed bands. A black tooling line is provided in order to indicate where images were overlaid. A Kruskal-Wallis test with a correction for multiple comparisons was performed by controlling the false discovery rate using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *, q < 0.05 compared to the control (NoLF); nat pLF, native porcine lactoferrin; rec pLF, recombinant porcine lactoferrin; M, MagicMark protein standard; L, Precision Plus Protein all blue.

The proteolytic effect of lactoferrin on flagellin and heat-labile enterotoxin.

Bovine LF and native and recombinant pLF degrade F4 and F18 fimbriae. To investigate if the protease activity of bLF, ovoTF, and pLF (native and recombinant) degrades LT, we performed Western blotting on supernatant of an ETEC strain secreting only LT (IMM01△STb) (Fig. 5A and B). Coincubating bLF, ovoTF, native pLF, and recombinant pLF with bacterial culture medium containing LT did not result in the degradation of the A or B subunits of LT (Fig. 5A and B). However, other virulence factors, such as flagellin, are also important for the motility of the pathogens. Similar to what was observed for FedA and FedF, all lactoferrins significantly degraded flagellin to a similar extent (Fig. 5C).

FIG 5.

The effect of lactoferrins on heat-labile enterotoxin and flagellin degradation. (A to C) The relative quantification of (A) heat-labile enterotoxin subunit A degradation (n = 3) with a representative Western blot, (B) heat-labile enterotoxin subunit B degradation (n = 3) with a representative Western blot, and (C) flagellin degradation (n = 4) with a representative Western blot. In panel B, no visible molecular ladder was present on the Western blot, so an overlay was created between the prestained ladder and the Western blot image in order to determine the molecular weights of the observed bands. A black tooling line is provided in order to indicate where images were overlaid. Data in the graphs are normalized to their respective controls (no LF) and presented as the mean ± SD. Lactoferrins at 1 mg/ml were used to assess degradation of heat-labile enterotoxin and flagellin. A Kruskal-Wallis test with a correction for multiple comparisons was performed by controlling the false discovery rate using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *, q < 0.05 compared to the control (in GraphPad Prism). nat pLF, native porcine lactoferrin; rec pLF, recombinant porcine lactoferrin; M, MagicMark protein standard.

Lactoferrin decreases the adhesion of ETEC to porcine small intestinal epithelial cells.

The degradation of fimbriae by lactoferrins could affect the adhesion of ETEC bacteria to the small intestinal epithelial cells. Hence, a cell attachment assay was performed using the porcine small intestinal epithelial cell line IPEC-J2 to assess the effect of bLF, ovoTF, native pLF, and recombinant pLF on the adhesion of F4⁺ ETEC to the small intestinal epithelium. The highest lactoferrin concentration (1 mg/ml) that did not affect bacterial growth (Fig. 2) was used to assess the effect on adhesion. As shown in Fig. 6, bLF, native pLF, and recombinant pLF decreased the adhesion of the F4⁺ ETEC strains GIS26, IMM01, G7, and H56 to IPEC-J2 monolayers (Fig. 6A to D). Although ovoTF did not degrade F4 fimbriae, it clearly decreased adhesion of the strains GIS26, G7, and H56, but not IMM01 (Fig. 6D). To ascertain that this decrease in adhesion is the result of its proteolytic activity, we first inhibited the proteolytic activity of LF by an irreversible interaction with PMSF. This inactivated LF was then used in the in vitro adhesion assay. PMSF-inactivated LF was unable to decrease the adhesion of G7 (Fig. 6E) and GIS26 (Fig. 6F) to IPEC-J2 cells, revealing that the proteolytic activity of lactoferrin is responsible for the observed decrease in adhesion.

FIG 6.

The relative adhesion of different ETEC strains to the IPEC-J2 cells or villi. (A to D) The relative adhesion of (A) O8:K87 (G7), (B) O149:K91 (GIS26), (C) O8:K87 (H56), and (D) O149:K91 (IMM01) to IPEC-J2 cells upon coincubation with LF. (E and F) The relative adhesion of (E) O8:K87 (G7) and (F) O149:K91 (GIS26) to IPEC-J2 cell upon coincubation with PMSF-inactivated LF. (G) The relative adhesion of F107/86 to isolated villi of pigs. Lactoferrins at 1 mg/ml were used to assess the adhesion of several F4⁺ and F18⁺ ETEC strains to IPEC-J2 cells and isolated villi, respectively, while a condition without lactoferrin was added as a control (NoLF). In panels A to D and G, data in the graphs are normalized to their respective controls (NoLF) and presented as the mean percentage ± SD (n = 4). In panels E and F, data in the graphs are normalized to their respective controls (NoLF) and presented as the mean percentage ± SD (n = 3). A Kruskal-Wallis test with a correction for multiple comparisons was performed by controlling the false discovery rate using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *, q < 0.05 compared to the control (NoLF); nat pLF, native porcine lactoferrin; rec pLF, recombinant porcine lactoferrin.

In addition, an in vitro villus adhesion assay was performed in order to assess the effect of the different LF on the adhesion of F18⁺ ETEC to small intestinal epithelial cells. Coincubating bLF, ovoTF, and pLF (native and recombinant) with strain F107/86 showed that LF also decreases the adhesion of a porcine F18⁺ ETEC strain to isolated villi (Fig. 6G).

DISCUSSION

The discovery and development of antibiotics has greatly reduced morbidity and mortality from bacterial infections. However, extensive use, and even misuse, of these antibiotics in large-scale intensive animal production systems has driven the rapid evolution and spread of antibiotic resistance (11, 35). In low- and middle-income countries, antibiotic resistance in pathogens of food-producing animals (pig and poultry) has increased in the last decade. AMR is expected to further increase as the global demand for animal protein rises in the coming decade and, with it, the use of antibiotics (36). This presents a major threat to the effective treatment of diseases caused by bacterial pathogens. Thus, alternatives for antibiotics in food-producing animals, such as pigs, are urgently needed. The antibacterial activity of lactoferrin for Gram-positive and Gram-negative bacteria has been widely documented both in vitro and in vivo (37–39). We previously demonstrated that human LF and bLF inhibited the growth of an E. coli O157:H7 strain and its attachment to intestinal epithelial cells, with bLF being significantly more effective (24). Furthermore, we showed that LF and ovoTF can inhibit attachment and cell entry of C. psittaci in chicken macrophages in vitro (28). Interestingly, ovoTF was significantly more effective than human or bovine LF. Also, in vivo, ovoTF efficiently prevented C. psittaci infection in experimentally infected specific-pathogen-free (SPF) turkeys (40) and on a commercial turkey farm (41). These results and those in the bovine system showed that LF of one species is more effective in that species and suggest that it affects the pathogen directly as well as through interaction with the host cells. Here, we wanted to assess if a similar species-dependent efficacy could be observed for pLF in comparison with bLF and ovoTF on porcine ETEC strains. Both native pLF, purified from milk (42), and recombinant pLF, produced using a workflow developed by our laboratory (31), were evaluated.

Bovine and human LF are known to inhibit the growth of several bacterial strains, including Streptococcus mutans, S. aureus, and E. coli (43–45). Here, we show that the different LF are able to inhibit growth of porcine ETEC strains at similar concentrations. In this study, we observed that only the highest concentrations of pLF and bLF (5 and 10 mg/ml) showed a significant growth inhibition. Bovine lactoferrin was slightly more effective than native and recombinant pLF in inhibiting the growth of the porcine ETEC strains. However, this did not result in a lower effective concentration, as both bLF and pLF inhibited ETEC growth at 10 and 5 mg/ml. Moreover, the growth inhibition by bLF and pLF, at these concentrations, remained significant even at 8 h postincubation. The latter was in contrast to the growth inhibition observed in E. coli O157:H7, where the growth was only significantly inhibited between 3 and 6 h postincubation (24). This indicates that the growth inhibition by bLF and pLF, at these high concentrations, might be attributed to a direct interaction of bLF and pLF with LPS, causing an increase in cell wall permeability and resulting in bacterial death (23, 46). Interestingly, the growth inhibition seems to be associated with the serotype of the bacteria (which has, as far as we know, not yet been reported), as similar growth inhibition patterns were observed for the O149 serotype strains GIS26 and IMM01 as well as the O8 strains G7 and H56. However, it was previously shown that the ability of LF to inhibit growth varied between strains (45).

It has been reported that bLF and human lactoferrin have proteolytic activity which is similar to that of serine proteases and can be reversibly or irreversibly inhibited by benzamidine or PMSF and by Pefabloc, respectively (26). This proteolytic activity permits them to degrade colonization factors of Haemophilus influenzae (47) and virulence factors associated with the type III secretion system of EHEC bacteria, such as EspA and EspB (24). Here, we demonstrated that there were no significant differences in proteolytic activity between bLF and native and recombinant pLF. All tested lactoferrins degrade the tip adhesion subunit FedF and the structural subunit FedA of F18 fimbriae. Furthermore, bovine LF and pLF degraded the F4 fimbrial major subunit FaeG, but not ovoTF. The degradation of F4 and F18 fimbriae by LF is meaningful, as these fimbriae mediate the attachment of ETEC to the small intestinal epithelium. We previously showed that the degradation of EspA and EspB by bLF and hLF was associated with a decreased adhesion of E. coli O157:H7 to the Caco-2 cell line (24). It has been reported that hLF can cleave H. influenzae IgA1 protease and the H. influenzae Hap adhesin in the arginine-rich regions RRSRRSVR and VRSRRAAR, respectively (25). The latter could explain why LF was unable to degrade LT, as these arginine-rich regions may not be present or may be shielded by its native structure. The capacity of LF to degrade bacterial virulence factors might be essential in the inhibition of bacterial adhesion to host cells. In the present study, no significant differences were observed between all tested lactoferrins when inhibiting the adherence of F4⁺ ETEC strains to IPEC-J2 cells. This corresponds to previous observations where hLF and bLF were shown to inhibit the adhesion of ETEC strains upon treatment with lactoferrin (48, 49). Furthermore, we also show that this decrease in adhesion is dependent on the proteolytic activity of LF. Although ovoTF was unable to degrade F4 fimbriae, it clearly decreased adhesion of the strains GIS26, G7, and H56, but not IMM01. This strain differs from the other strains in that it does not produce flagellin. Flagellin might be involved in the adhesion of ETEC bacteria to IPEC-J2 cells. However, the role of flagellin in ETEC colonization of the small intestinal mucosa remains unclear (50). Interestingly, identical results were obtained for recombinant and native pLF for growth inhibition, protease activity, and inhibition of adhesion. This showed that the efficacy of recombinant-produced lactoferrin is similar to that of the native pLF.

In conclusion, the antibacterial and proteolytic activity of lactoferrin resulted in a significant growth inhibition and degradation of ETEC-associated virulence factors, respectively. Furthermore, the results from this study indicate that the proteolytic activity of lactoferrin is responsible for the observed decrease in the in vitro attachment of ETEC to intestinal epithelial cell culture (IPEC-J2). Further studies are needed to evaluate the impact of in-feed lactoferrin on ETEC pathogens in piglets. Our findings support the further development of lactoferrin as a strategy to provide protection against colonization and infection and reduce antibiotic use in livestock species.

MATERIALS AND METHODS

Cell cultures.

IPEC-J2 is a nontransformed porcine intestinal epithelial cell line derived from the jejunal epithelium of a neonatal, unsuckled piglet. The cell line is maintained as a continuous culture in Dulbecco’s modified Eagle medium/F12 (DMEM/F12 1:1; Gibco, Merelbeke, Belgium), supplemented with 5% fetal calf serum (FCS), 2 mM l-glutamine (Gibco), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (ITS, Gibco), 100 U/ml penicillin and 100 μg/ml penicillin-streptomycin (P/S; Gibco), and 5 ng/ml epidermal growth factor (EGF; Invitrogen, Merelbeke, Belgium) at 37°C and 5% CO₂ in a humidified atmosphere. These undifferentiated cells reach confluence after 3 to 4 days, after which the cell line is subcultured with phosphate-buffered saline (PBS) containing 0.25% trypsin (Gibco), as well as 100 U/ml penicillin and 100 μg/ml streptomycin (P/S; Gibco).

The Chinese hamster ovary (CHO) cell line is derived from the Chinese hamster ovary and has become a stable source of cells due to their robust growth as adherent or suspension cells. The cell line is maintained as a continuous culture in Ham’s F12 (Gibco, Merelbeke, Belgium) supplemented with 10% FCS plus 100 U/ml penicillin and 100 μg/ml streptomycin (P/S; Gibco) at 37°C and 5% CO₂ in a humidified atmosphere. These cells reach confluence after 4 to 5 days, after which the cell line is subcultured with PBS containing 0.25% trypsin (Gibco) plus 100 U/ml penicillin and 100 μg/ml streptomycin (P/S; Gibco).

The highest-producing clone was adapted to suspension culture growth as described elsewhere (51). The suspension medium (EX-CELL ACF CHO; Sigma) was supplemented with 6 mM l-glutamine, 5% FCS, 1% P/S, and 0.15 mg/ml G418. Suspension-adapted cells were seeded at a density of 5.0 × 10⁵ cells/ml and grown in 200 ml medium in 500-ml shaker flasks at 37°C and 150 rpm without CO₂. When the maximal cell density was reached (3.0 × 10⁶ cells/ml), the cells were first expanded to 400 ml and then 2 liters of medium in 1- and 5-liter shaker flasks, respectively, at a seeding density of 5.0 × 10⁵ cells/ml for recombinant pLF production. When the cells reached their maximal density (3.0 × 10⁶ cells/ml) in the 5-liter shaker flasks, the temperature was shifted to 30°C for optimal protein production. The cell culture supernatant was collected after 10 days, when the cell viability dropped below 90%.

Transfection of CHO cells with a pcDNA3.1+N-eGFP-pLF vector.

The construct, generated by Genscript, contains the pLF sequence as previously described (52) and was cloned into the pcDNA3.1+N-eGFP vector. The pcDNA3.1+N-eGFP vector containing the pLF construct was then used to transfect CHO cells. CHO cells were first grown to 60 to 80% confluence and subsequently transfected by adding jetPRIME buffer (Polyplus-transfection) containing 0.5 μg of the pLF construct DNA to the medium. The medium was replaced after 4 h, and the transfected CHO cells were then grown under an optimized Geneticin selection (1 mg/ml). The Geneticin-resistant CHO cells were first checked for GFP expression using a fluorescence microscope and were subsequently trypsinized, and the 4% highest-GFP-expressing cells were then single cell sorted into 96-well plates using the BD FACSARIA III cell sorter (BD Biosciences). These cells were then grown to 100% confluence. Fast-growing clones with a homogenous high expression of GFP as evaluated by live-cell microscopy (an Olympus Cell̂M imaging system) were selected and cultured. The clone producing the greatest amount of pLF, as determined by Western blotting, was selected and expanded as detailed above for pLF production.

Purification of ETEC virulence factors.

F4 fimbriae were purified from the ETEC strain GIS26 as previously described (53). Briefly, the bacteria were cultured in tryptone soy broth (TSB; Difco Laboratories, Biotrading, Bierbeek, Belgium) at 37°C for 18 h, collected by centrifugation, and washed in sterile PBS. Subsequently, F4 fimbriae were isolated by mechanical shearing of the bacterial suspension followed by centrifugation to remove the remaining bacteria. The fimbriae were precipitated with ammonium sulfate (40% saturation), and the pellet was dissolved in PBS and dialyzed overnight against PBS at 4°C.

F18 fimbriae were purified from the F18⁺ strain F107/86 as previously described (54). Briefly, the bacteria were cultured in TSB at 37°C for 18 h, collected by centrifugation, and washed in sterile PBS. The F18 fimbriae were isolated by mechanical shearing of the bacterial suspension, centrifuged for 20 min at 1,251 × g at 4°C, and subsequently precipitated with ammonium sulfate (20% saturation). The pellet was dissolved in PBS and dialyzed overnight against PBS at 4°C.

Flagellin was isolated from strain GIS26△faeG using the same protocol as used to purify F4 fimbriae. The protein concentration of the purified proteins was determined by the bicinchoninic acid (BCA) reaction (Pierce BCA protein assay kit) with bovine serum albumin as a standard, and their purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%) and Coomassie staining.

Purification of recombinant and native porcine LF.

The suspension medium containing recombinant pLF was run over a Histrap HP column (GE Healthcare) overnight at 4°C at a flow rate of 1 ml/min. The column was washed using a washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4) to remove nonspecific bound proteins. A stepwise gradient of the elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4) was used, and the eluate was collected. The elution fractions were dialyzed against PBS overnight at 4°C, after which the protein concentration was determined using BCA (Pierce BCA protein assay kit). The purity of the elution fractions was determined by silver staining (Pierce silver stain kit) following the manufacturer’s specifications.

Raw milk samples were collected from a pig farm and stored at –20°C. The pLF was isolated from the milk as previously described (42). Briefly, the milk samples were centrifuged for 30 min at 4,000 × g and 4°C. The fat layer was removed, the pH was decreased to 4.6 to precipitate casein, and the sample was subsequently centrifuged at 4,000 × g for 30 min at 4°C. The supernatant was neutralized to pH 6.8 and subsequently run over a HiTrap heparin HP column (GE Healthcare) at room temperature at a flow rate of 1 ml/min. The column was washed using a washing buffer (10 mM sodium phosphate, pH 7). A stepwise gradient of the elution buffer (10 mM sodium phosphate, 2 M NaCl, pH 7) was used, and the eluate was collected. The elution fractions were dialyzed against PBS overnight at 4°C. The protein concentration and purity of the fractions were determined as described above.

Iron saturation.

Hololactoferrin was prepared by incubating 50 mg/ml lactoferrin solution (in 50 mM Tris-HCl) and 150 mM NaCl (pH 7.4) with ferric nitrate salt Fe(NO₃)₃ (9.9 mM) in the presence of nitrilotriacetic acid (8.5 mM) in water adjusted to pH 7.0 with sodium bicarbonate. After shaking the solutions overnight at 4°C, excess iron was removed by dialysis (Slide-A-Lyzer dialysis cassettes; Thermo Fisher) against the same buffer solution without ferric salts for 24 h and against water for another 24 h. The iron saturation was determined by measuring the A₂₈₀/A₄₆₆ ratios, using the high-throughput method, as previously described (32).

Western blotting.

To assess if lactoferrin can degrade F4 fimbriae, F18 fimbriae, heat-labile enterotoxin, and flagellin, we incubated purified F4 and F18 fimbriae, bacterial medium containing LT, and flagellin at a final concentration of 20 μg/ml in DMEM containing different concentrations of bLF (Ingredia Nutritional, France), ovoTF (C0755; Sigma), and native and recombinant pLF for 5 h at 37°C as previously described (24). We also analyzed the degradation of denatured F4 fimbriae by boiling purified F4 fimbriae and then incubating these fimbriae in DMEM containing 1 mg/ml bLF, ovoTF, or pLF. The proteolytic activity of LF was inhibited by incubating LF (1 mg/ml) with 0.5 mM PMSF in DMEM for 1 h at room temperature (RT). Purified F4 and F18 fimbriae at a final concentration of 20 μg/ml were subsequently added to the PMSF-inactivated LF and incubated for 5 h at 37°C. Laemmli buffer (6×) with β-mercapto-ethanol was added, and the samples were incubated for 5 min at 95° to be further analyzed by SDS-PAGE and Western blotting.

Proteins were first separated with SDS-PAGE (12%) and were then blotted for 120 min at 80 V onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with a 10% milk solution, washed with PBS-Tween 20 (0.1%), and then incubated with a mouse monoclonal antibody against FaeG (clone IMM01; in-house [55]), FedA (clone IMM02; in-house [56]), or FedF (clone IMM03; in-house [56]) and rabbit polyclonal antiserum against flagellin (in-house) or LT (ab188541; Abcam). The PVDF membrane was then incubated with an HRP-labeled secondary antibody (goat anti-mouse IgG-HRP, PO217 Dako, or swine anti-rabbit IgG-HRP, PO0447 Dako), and the protein was subsequently detected by chemiluminescence using the Pierce ECL Western blotting substrate detection kit (Thermo Scientific). Both the primary and secondary antibodies were diluted 1/1,000 in 10% milk. The relative band intensities were determined using Image Lab software (Bio-Rad).

Growth inhibition assay.

To assess if lactoferrin can inhibit growth of E. coli cultures, we incubated different E. coli strains (Table 2) in a concentration series of ovoTF, bLF, pLF, and rpLF in Luria-Bertani (LB) broth. These ETEC strains were prepared by inoculating a colony into a 10-ml tube containing LB and incubating the tube at 37°C for 12 to 18 h with shaking (180 rpm). Overnight ETEC cultures were pelleted by centrifugation (11,337 × g, 5 min) and reconstituted in 1 ml of LB medium. Bacteria (10⁷ CFU/ml as determined by the optical density at 600 nm [OD₆₀₀], 200 μl/well) were incubated in a 96-well plate (Greiner Bio-One) at 37°C for 8 h in LB broth supplemented with different concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, and 10 mg/ml) of bLF or ovoTF. Selected concentrations were within the physiological range. Bacterial growth was monitored spectrophotometrically (OD₅₉₅) hourly for 8 subsequent hours.

Cell adhesion assay.

The attachment of F4⁺ E. coli in the presence and absence of lactoferrins was determined using the porcine small intestinal epithelial cell line IPEC-J2 as previously described (24). The effect of lactoferrins on bacterial adhesion was assessed using the highest lactoferrin concentration (1 mg/ml) that did not inhibit bacterial growth, as determined in the growth inhibition assay. The proteolytic activity of LF was inhibited by incubating 10 mg/ml LF (in PBS) with 1 mM PMSF for 1 h at RT and then dialyzing overnight against PBS. The LF samples were subsequently filtered (0.45 μm), the protein concentration was determined using BCA, and then the PMSF-inactivated LF was added to IPEC-J2 culture medium without antibiotics at a final concentration of 1 mg/ml. E. coli cultures were prepared by inoculating a colony into a 10-ml tube containing LB broth and incubating the tube at 37°C for 12 to 18 h with shaking (180 rpm). Overnight cultures were pelleted by centrifugation (11,337 × g, 5 min) and reconstituted in 1 ml of DMEM. IPEC-J2 monolayers were seeded in a 24-well plate (10⁵ cells per well) and then grown to confluence in DMEM/F12 medium without antibiotics; they were subsequently infected with E. coli (MOI, 10) in the presence or absence of LF or PMSF-inactivated LF and further incubated for 2.5 h at 37°C and 5% CO₂. After incubation, nonadherent bacteria were removed by three PBS washes. IPEC-J2 cells were lysed by adding 0.25% trypsin for 30 min (37°C) and vigorously pipetting. Adherent bacteria were enumerated by spread plating appropriate 10-fold serial dilutions onto LB medium plates in triplicate. The LB medium plates were incubated at 37°C for 24 h, and the CFU were enumerated. Values were calculated relative to those obtained for the condition without LF.

Villus adhesion assay.

The in vitro villus adhesion assay was performed as previously described (57). The jejunal villi of four different pigs were first washed with Krebs buffer and subsequently suspended in PBS with 1% d-mannose, which prevents adhesion of E. coli by type 1 pili. Then, 3.2 × 10⁷ bacteria were added to 450 μl of PBS containing 1% d-mannose, villi, and lactoferrin (1 mg/ml). These samples were then incubated for 45 min at room temperature while rotating and were subsequently analyzed by phase contrast at a magnification of ×400. The number of adherent bacteria was determined by counting the bacteria adhering to a 50-μm brush border length in 20 different places.

Data analysis.

Statistical analysis was performed using GraphPad Prism 7. The bacterial growth was analyzed using a 2-way analysis of variance (ANOVA), where a P value of <0.05 was considered significant. The assumption of normality was determined using the Shapiro-Wilk normality test. The degradation of virulence factors was determined by measuring the band intensity of the target protein for each condition using Image Lab software (Bio-Rad). The obtained values were then normalized to the value of the condition without lactoferrin. A similar strategy was used to analyze the attachment of bacteria to IPEC-J2 cells, where the number of adherent bacteria was normalized to their respective controls and represented as a percentage. The degradation of virulence factors by lactoferrin and attachment to IPEC-J2 cells were analyzed using the nonparametric Kruskal-Wallis test with a correction for multiple comparisons by controlling the false discovery rate (FDR) using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. The effects of LF on degradation and adhesion were considered significant when q was <0.05, where the q value is a P value that has been adjusted for the FDR.