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Serum high-density lipoprotein (HDL) inhibits in vitro enterohemolysin (EHly) activity produced by enteropathogenic Escherichia coli

Patricia M.S. Figueirêdo, Cleide F. Catani, Tomomasa Yano
DOI: http://dx.doi.org/10.1016/S0928-8244(03)00125-1 53-57 First published online: 1 August 2003


Enterohemolysin (EHly) produced by Escherichia coli shows hemolytic activity towards washed erythrocytes from different animal species on blood agar plates. It has been shown recently that EHly activity is inhibited by normal mammalian serum and by cholesterol in vitro. Plasma lipoproteins can interact with bacterial toxins, such as endotoxin, to reduce their toxicity. In this work, we examine the ability of human purified chylomicrons, very low-density lipoproteins, intermediate-density, low-density and high-density lipoproteins, to inhibit the hemolytic activity of EHly. Our results show that these lipoproteins are hemolysin inactivators, and that high-density lipoprotein is the most potent inhibitor of enterohemolytic activity.

  • Escherichia coli
  • Enterohemolysin
  • Inhibition
  • Lipoprotein

1 Introduction

Hemolysins are produced by many Gram-positive and Gram-negative bacteria. They act by disturbing the structure and function of membranes. This damage may result in lysis and cell death [1]. Some of these toxins are potential virulence factors that contribute substantially to bacterial pathogenicity. The most thoroughly studied cytolysin of Gram-negative bacteria is the α-hemolysin among the different Escherichia coli hemolysins [2,3,4], which is an important virulence factor [5]. Some strains of enteropathogenic E. coli produce enterohemolysin (EHly), which is associated with enteric disease [3]. In contrast to α-hemolysin, EHly is not detected in E. coli culture supernatant. In addition, EHly activity is detected only on blood agar plates with washed erythrocytes suggesting that there is a factor in serum that inhibits the hemolytic activity of this toxin. In a previous report (Figueirêdo, Catani and Yano, submitted for publication), we found that enterohemolytic activity was inhibited by cholesterol with a similar concentration to that of normal serum.

Lipoproteins are aggregates of lipids and proteins, responsible for the transportation of lipids in organisms. They are organized in a manner that enables them to carry out their functions adequately. The lipoproteins are classified in five major classes according to their density: chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL); the greater the lipid:protein ratio the lower their density. The organization of lipoprotein contains a core, which consists of apolar lipids (cholesterol ester and triglycerides), and an envelope that contains the amphiphilic lipids (phospholipids and cholesterol) and the specific apoproteins for each particle of lipoprotein. Their lipid and protein composition define their metabolic behavior and biological activities.

Inflammation and infection by microorganisms can disturb lipoprotein metabolism and alter the plasma concentrations of lipids and lipoproteins [6]. Such disturbances in lipid metabolism are part of the host's defense because the immune response is tightly linked to the metabolic response [7]. Indeed, lipid particles, principally HDL, may be an integral part of the humoral detoxification mechanism [8]. In this study we investigated the ability of lipoproteins to inhibit the hemolytic activity of EHly.

2 Materials and methods

2.1 Bacterial strains

The strains used in this work were standard E. coli C3888, kindly supplied by Dr. L. Beutin (Robert Koch Institute, Germany), and E. coli B3, which was used as a negative control.

2.2 Purification of EHly

E. coli strain C3888 was grown in trypticase soy broth (TSB) with 12 µg of ethylenediamine hydroxyphenylacetic acid (EDDA) ml−1 and incubated for 22 h at 37°C, with constant shaking at 150 rpm (New Brunswick Scientific). Cultures were centrifuged at 10,000×g for 15 min at 4°C and the cell pellet was suspended in 100 ml of 10 mM Tris–HCl buffer, pH 7.6, and disrupted by ultrasonication (10 cycles, 10 s, 50% energy pulse) in a cell disrupter (Vibra Cell) followed by centrifugation for 20 min at 12,000×g. The resulting supernatant was used as a source of crude EHly. This EHly was fractionated with ammonium sulfate (60% saturation) at 4°C and the precipitate was collected by centrifugation and dissolved in 10 mM Tris–HCl, pH 7.6. After dialysis, the resulting material was applied to a DEAE Sepharose Fast Flow column (Amersham Pharmacia Biotech). Fractions with hemolytic activity were collected and concentrated by membrane ultrafiltration (PM-50 Diaflo, Amicon). The concentrated hemolysin was applied to a Superdex 200 column (Amersham Pharmacia). The material obtained in this chromatography represented partially purified EHly and was used in tests presented in this paper. As the final step in purification, the material (0.23 mg) from the preceding step was applied to a fast protein liquid chromatography HR 5/5 Mono Q anion-exchange column (Pharmacia LKB) (Figueirêdo, Catani and Yano, submitted for publication).

2.3 Quantitation of hemolytic activity by microplate method

Horse erythrocytes were washed three times with 5 mM phosphate-buffered saline (PBS), pH 7.2, and resuspended to a concentration of 1%. An aliquot (50 µl) of the erythrocyte suspension was added to 50 µl of serially diluted (two-fold dilutions) crude EHly in 96-well microtiter plates. The plates were then incubated for 60 min at 37°C. The hemolytic titer was defined as the reciprocal of the highest dilution of toxin that produced approximately 50% hemolysis. This reciprocal value indicates the number of hemolytic units (HU) in the undiluted toxin [9].

2.4 Inhibition of enterohemolytic activity by normal serum

The inhibition of crude EHly by normal serum was examined by mixing equal volumes of EHly with serially diluted (two-fold dilutions) sera from oxen, mice and humans. An initial incubation at 37°C for 60 min was followed by further incubation at 4°C for 18 h. The residual activity of EHly was assayed by adding 100 µl of horse erythrocyte suspension to the microplate followed by incubation at 37°C for 1 h.

2.5 Hemolytic activity in the presence of cholesterol

The influence of cholesterol on hemolytic activity was tested as described by Geoffroy et al. [10]. An alcoholic solution of cholesterol (10 mg ml−1) was diluted serially (two-fold dilutions) in 96-well microtiter plates to a concentration of 50 µg ml−1, and the cholesterol was incubated for 60 min at 37°C with aliquots of EHly followed by 18 h at 4°C. The hemolytic activity was detected by incubating the above mixture with a 1% suspension of erythrocytes for 60 min at 37°C.

2.6 Purification of major lipoproteins

HDL and other lipoproteins were purified from fresh human sera by density-gradient ultracentrifugation [11]. Stock solutions of lipoproteins were dialyzed extensively against 20 mM Tris–HCl, pH 7.5, containing 140 mM NaCl and 1 mM EDTA, then passed through a 0.22-µm filter and stored sterile in the dark at 4°C for 15 days. The lipoprotein concentration was determined by a protein assay (Bio-Rad, Richmond, CA, USA) and a total cholesterol test kit (Sigma, St. Louis, MO, USA).

2.7 Inhibition of the hemolytic activity of EHly by lipoprotein

The assays were done at 37°C in 5 mM PBS, pH 7.2, with a fixed lipoprotein concentration and different crude EHly concentrations, or with varying amounts of lipoprotein and a constant EHly concentration. After incubation at 37°C for 60 min followed by another incubation at 4°C for 18 h, the hemolytic activity of EHly was assayed by adding 100 µl of horse erythrocyte suspension to the microplate wells. This was followed by another incubation at 37°C for 1 h, as described in Section 2.3.

3 Results

3.1 Production and purification of EHly and quantitation of hemolytic activity

Crude EHly prepared as described above was applied to a DEAE Fast Flow column equilibrated with 10 mM Tris–HCl, pH 7.6. The fractions with hemolytic activity were eluted with 0.1 M NaCl, followed by concentration with an Amicon XM 50 membrane (Amicon) and gel filtration on Superdex 200. Hemolytic fractions from the gel filtration step were applied to a Mono Q column. The EHly was eluted with 0.15 M NaCl and purified about 100-fold with a recovery of 0.06%. But the storage of eluted material at 4°C overnight led to complete loss of hemolytic activity (Figueirêdo, Catani and Yano, submitted for publication), so this preparation was not used in the present work. The recoveries and yields from a typical purification are summarized in Table 1.

View this table:
Table 1

Purification of E. coli EHly

FractionTotal volume (ml)Total protein (mg)Total activity (HU)Specific activity (HU mg−1)Relative activity
Ammonium sulfate (60%)204212.5×10459.702.1
DEAE Fast Flow2.52.448.0×102327.911.7
Superdex 2001.00.231.6×102695.624.8
Mono Q0.50.0070.2×1022857.0101.8
  • Sonicated material of 2.5 l of culture in TSB with EDDA.

3.2 Inhibition of hemolytic activity by normal mammalian sera and cholesterol

Normal mammalian sera inhibited the hemolytic activity of EHly on washed erythrocytes, as shown in Table 2.1.

View this table:
Table 2.1

Inhibition of the hemolytic activity of EHly by normal mammalian sera

Serum (mg ml−1)TiterMIC (µg ml−1)
Human (2.25)1/10122
Ox (1.7)1/10123
Mouse (0.97)1/2563
  • Pool of normal sera with total cholesterol concentration in parentheses.

  • Last serum dilution that showed 100% inhibition of EHly.

  • Minimum cholesterol concentration that showed 100% inhibition of EHly.

Concentrations of cholesterol ≥2.5 mg ml−1 totally inhibited hemolysis, whereas concentrations of 1.25–2.3 mg ml−1 produced only partial inhibition (Table 2.2).

View this table:
Table 2.2

Hemolytic activity in the presence of cholesterol

Cholesterol (mg ml−1)Hemolysis
  • Hemolysis 100%.

  • Hemolysis 50%.

  • Inhibition of hemolysis.

3.3 Inhibition of the hemolytic activity of EHly by lipoprotein

Total plasma lipoproteins were separated into the main lipoprotein classes by differential density ultracentrifugation under the conditions described above. This separation was dependent only on the densities of the lipoprotein species so separations were quite distinct (Table 3). Among the lipoprotein fractions tested for inhibition of hemolytic activity of EHly, HDL showed the highest titer of inhibition (Table 3), although all classes of lipoproteins inhibited the hemolytic activity to some extent.

View this table:
Table 3

Distribution of lipoprotein cholesterol in the pool of human sera and inhibition of EHly activity

DensityDesignationCholesterol (mg ml−1)TiterMIC (µg ml−1)
  • Density adjusted with solid potassium bromide (KBr) and salt solution (NaCl).

  • Last dilution that showed 100% inhibition of EHly activity.

  • Minimum cholesterol concentration that showed 100% inhibition activity of EHly.

  • Pellet (bottom of the tube)=other plasma proteins.

4 Discussion

Recently it has been suggested that EHly alters and disrupts cell membranes by a detergent-like mechanism [12]. Detergents form a special group of lipids that are used to solubilize membranes [13], and act by forming thermodynamically stable colloidal aggregates or micelles [14] that may be asymmetrically distributed between the two leaflets of the membrane lipid bilayer. Interactions between lipids and membrane proteins may further affect the state of the lipids causing disruption of the membranes into mixed micelles or even separation of the lipid and protein constituents.

The results obtained from the assay of inhibition of EHly activity by human, ox and mouse sera (Table 2.1) justify the necessity of using washed erythrocytes for the detection of EHly activity as described by Beutin et al. [3], because mammalian sera inhibit the biological activity of EHly in vitro and the main cause may be the presence of cholesterol. Cholesterol, which inhibits the hemolytic activity of EHly in vitro (Table 2.2), is located in cell membranes and occurs in mammalian sera as a component of lipoproteins, which are responsible for the transportation of lipids in the organism. Lipoproteins can be separated into five different fractions according to their density: chylomicrons, VLDL, IDL, LDL, and HDL. All of these lipoproteins have lower densities than other plasma proteins and can thus be separated and purified by density-gradient ultracentrifugation (Table 3). Results demonstrated in Table 3 show that all lipoproteins purified from human serum inhibited the hemolytic activity of EHly, and HDL showed the highest titer of inhibition whereas the other plasma proteins (pellet) had no activity. This finding indicates that lipoproteins are the inhibitory factor of EHly activity in normal serum, particularly HDL.

Some research reports have pointed to the relation between infection and inflammation and alterations in cholesterol metabolism. In addition to changes in circulating levels of cholesterol, it is proposed that HDL mediates the removal of cholesterol from peripheral cells and returning it for elimination, a pathway known as reverse cholesterol transport [15]. This may emphasize the importance of HDL acting as a vehicle for the reverse transport of lipids, providing protection and inactivation of biologically active lipids, such as endotoxin (lipopolysaccharide, LPS) in vivo [16] and in vitro [8,17].

LPS is a lipopolysaccharide that is released from the outer membrane of Gram-negative bacteria responsible for hemodynamic, hematological, and metabolic changes, observed during severe infections [8,18]. In plasma LPS can form complexes with lipoproteins, mainly HDL, and some researchers suggest a simple leaflet insertion model for binding and neutralization of LPS by phospholipid on the surface of HDL [16,19,20]. Our results suggest that the mechanism by which lipoproteins inhibit the activity of EHly may be similar to the inactivation of LPS but occurs by cholesterol on the surface of HDL and other lipoproteins.

We show here that lipoproteins, particularly HDL, inhibited EHly activity in vitro. Recent evidence that HDL is involved in acute and chronic infections suggests that plasma lipoproteins may represent another line of defense during infectious or inflammatory processes [21].

The mechanism by which lipoproteins inhibit the EHly activity requires further study in order to explain the physiological relevance of HDL. Other lipoproteins may act like acute-phase response proteins and the effects of EHly produced by enteropathogenic E. coli.


This work was supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo). We thank Dr. Helena Coutinho Franco de Oliveira for helpful discussion and the Clinical Laboratory of Pathology of H.C. (Hospital das Clínicas) of Unicamp, Campinas for supplying the pool of human sera.


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