Modulation of host cell membrane fluidity: a novel mechanism for preventing bacterial adhesion

Arif Ismaili1,2, Jonathan B. Meddings3, Samuel Ratnam4, and Philip M. Sherman1,2,5

1 Division of Gastroenterology and Nutrition, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8; 3 Gastrointestinal Research Group, University of Calgary, Calgary, Alberta T2N 4N1; 4 Newfoundland and Labrador Public Health Laboratory, St. John's, Newfoundland A1B 3T2; and Departments of 5 Pediatrics and 2 Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adhesion of bacterial enteropathogens to host mucosal surfaces is a critical primary step in the pathogenesis of diarrheal disease. We investigated the effects of altering the physical properties of eukaryotic cells on bacterial adhesion with the use of a series of three structurally dissimilar membrane fluidizers and several Escherichia coli as test strains. Lipid fluidity of the cell plasma membrane was measured by steady-state fluorescence anisotropy employing the probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene. There was a dose-dependent and reversible inhibition of bacterial adhesion with increasing membrane fluidity. Time course experiments indicated that increasing membrane fluidity during the early stages of bacterial adhesion was essential for inhibition of attachment. None of the fluidizers affected the viability of either eukaryotic or prokaryotic cells. These findings demonstrate, for the first time, that changes in plasma membrane physical properties of epithelial cells can prevent microbial adhesion. This also suggests that altering the membrane properties of host cells could form a basis for novel strategies to prevent bacterial adhesion during infection in vivo.

lipid fluidity; Escherichia coli; O157:H7; O127:H6


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADHESION OF MICROORGANISMS to host cells is a pivotal step in pathogenesis of disease (3). In certain situations, attachment is a prerequisite for invasion of the microbe into the host cytoplasm, and in other cases binding allows the organisms to be in close proximity to the host cells, thereby enhancing the delivery of their toxic products (12). Recently, the induction of signal-transduction cascades in the cytosol of an infected eukaryotic cell after binding of bacteria to host cell receptors has come to be recognized as a novel mechanism by which prokaryotes promote colonization (7, 30).

Bacterial binding to host cells is generally the result of a specific interaction between a surface ligand expressed by the prokaryote and a eukaryotic plasma membrane receptor (30). The conformation of membrane receptors can be altered by changing the physical properties of the plasma membrane (28). One way of influencing the physical properties of membranes is to change membrane fluidity. Previous studies have reported that binding of soluble mediators to surface receptors can be modulated by altering the membrane fluidity of target cells (2, 23, 27). Heron et al. (13) showed that fluidization of membrane lipids by treatment with linoleic acid causes a decrease in the binding of serotonin. Similarly, changing the fluidity of mouse hepatocyte membranes reduces the binding of prolactin (8). Changes in membrane fluidity not only reduce the affinity of binding of the chemotactic peptide formyl-Met-Leu-Phe to its membrane receptor but the downstream signal transduction events that mediate the varied biological activities of formyl-Met-Leu-Phe are also affected (31).

We carried out a study to determine whether the binding of pathogenic bacteria could be similarly influenced by changes in the plasma membrane fluidity of host cells. The study findings provide the first evidence that bacterial adhesion to host cells in vitro can be prevented by increasing the membrane fluidity of target cells with plasma membrane-mobilizing agents.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane Fluidizers

Known membrane fluidizing agents employed to increase the membrane fluidity of epithelial cells included hexanol (22), benzyl alcohol (22), and the fatty acid-like compound 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) (15) (Sigma Chemical, St. Louis, MO). Methanol (BDH, Toronto, ON, Canada) was employed as a negative control (22).

Bacteria and Growth Conditions

The attaching and effacing enteropathogenic Escherichia coli (EPEC) strain E2348/69 (serotype O127:H6) was used throughout the study (18). As a control, parallel experiments were carried out with four other E. coli strains adhering to epithelial cells by distinct mechanisms. These strains included attaching and effacing Shiga toxin-producing E. coli strain CL56 (serotype O157:H7), nonattaching and effacing Shiga toxin-producing E. coli strain CL15 (serotype O113:H21), enterotoxigenic E. coli strain H10407 (serotype O78:H11), and enteroinvasive E. coli strain CL114 (serotype O164:H-). E. coli were cultured in static, nonaerated Penassay broth (Difco Laboratories, Detroit, MI) overnight at 37°C. Bacteria were harvested from the broth culture by centrifugation at 2,500 g for 15 min followed by resuspension of the pellet in PBS (pH 7.4) to a concentration of 1 × 1010 colony forming U/ml.

Eukaryotic Cell Culture

The human epithelial cell line HEp-2 (CCL-23 from American Type Culture Collection, Manassas, VA), widely used as a model system to study the adhesion properties of human bacterial enteropathogens (11, 18), was used in this study. Cells were cultivated in minimum essential medium (Life Technologies, Grand Island, NY) supplemented with 15% FCS (Cansera International, Rexdale, ON, Canada), 0.5% glutamine, 0.1% sodium bicarbonate, 2% penicillin-streptomycin, and 1% amphotericin B (all from Life Technologies) at 37°C in 5% CO2.

Bacterial Adhesion Assays

Visual examination of Giemsa-stained HEp-2 cells. HEp-2 cells were grown overnight to subconfluence in LabTek chamber slides (Miles Scientific, Naperville, IL). The cells were washed free of the antibiotic-containing medium. An antibiotic-free medium containing the fluidizers (sonicated directly into the medium for 20 s) was then added to HEp-2 cells. The cells were infected with 5 × 107 bacteria in 0.005 ml sterile PBS for 3 h at 37°C. The fluidizers were left in 2 ml of tissue culture medium for the entire period of infection. Cells were then washed six times with PBS to remove nonadherent bacteria, fixed in 70% methanol for 10 min at 25°C, and stained with Giemsa (Fisher Scientific, Pittsburgh, PA) for 15 min at 25°C. After washing with distilled water to remove excess stain, the slides were examined under bright-field microscopy (Leitz Dialux 22, Leica Canada, Willowdale, ON, Canada).

Quantitative bacterial adhesion assay. HEp-2 cells were grown in 12-well culture plates (Costar, Cambridge, MA) to confluence and infected with 1 × 109 E2348/69 in 0.01 ml broth under the same conditions as described above. After cells were washed to remove nonadherent bacteria, the HEp-2 cells were detached from culture plates by using 0.25% trypsin (Life Technologies). After centrifugation and lysis of HEp-2 cells in distilled water containing in 0.1% BSA, to free the adherent bacteria on eukaryotic cells, serial 10-fold dilutions were plated onto MacConkey agar for viability counts. Results were expressed as the percent inhibition of adhesion observed in cells treated with membrane fluidizers compared with the control cells to which no fluidizer was added.

Membrane Fluidity Measurements by Steady-State Fluorescence Anisotropy

The fluidity of plasma membranes of whole living epithelial cells was measured according to the method previously described (16). The fluorescent probe, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) (Molecular Probes, Eugene, OR), was stored in the dark at room temperature as a 2 mM stock solution in DMSO. On the day of the experiment, a TMA-DPH labeling solution was made by diluting the stock solution in 4,000 volumes of a buffer containing (in mM) 137 NaCl, 2.7 KCl, 1 MgCl2, 0.4 NaH2PO4 5.6 glucose, and 10 HEPES (pH 7.4) (16). HEp-2 cells were labeled by mixing approximately 1 × 105 cells suspended in 1 ml of buffer with 1 ml of the labeling solution in a cuvette containing an additional 1 ml of buffer. A corresponding blank was also prepared in which the labeling solution was replaced with buffer. After 60 s to allow the probe to incorporate into the plasma membranes of HEp-2 cells, samples were read every 5 min for a period of 30 min (see RESULTS for details).

To evaluate the effects of hexanol and benzyl alcohol on membrane fluidity of HEp-2 cells, each of the fluidizers was added to the labeled cell suspension after the first reading was recorded. This time point then served as t = 0 min, with subsequent readings taken at 5-min intervals for a period of 20 min.

The steady-state anisotropy parameter (rs) for TMA-DPH was measured using an SLM 4800C spectrofluorometer (SLM/Aminco, Urbana, IL). The value of rs is directly related to the extent or range of the probe motion, which in turn is dependent on the packing of the surrounding lipids (29). The value of rs is inversely proportional to membrane fluidity; that is, a decrease in rs is indicative of an increase in membrane fluidity (15). The TMA-DPH probe was excited at a wavelength of 340 nm, and the emission wavelength was set at 420 nm. Samples were then excited alternately with vertically or horizontally polarized light, and the intensity of the emitted light polarized vertically (lv) and horizontally (lh) with respect to the exciting light measured simultaneously. As a result, the change in the direction of the light emitted by the probe is given as the steady-state anisotropy parameter, rs
r<SUB>s</SUB> = l<SUB>v</SUB> − l<SUB>h</SUB>/l<SUB>v</SUB> + 2l<SUB>h</SUB>
Total fluorescence intensity is provided by the denominator of this equation (16).

Data were calculated by reading alternately from the test sample and the blank in order to correct for scattered light (16). At each time interval, each sample was measured seven times. The procedure was repeated for at least four separate experiments.

Viability of Eukaryotic and Prokaryotic Cells

HEp-2 cell monolayers were incubated with each of the three fluidizers for 3 h at 37°C. After washing in PBS, the cells were detached from plastic surface by incubation with 0.25% trypsin for 5 min at 37°C, pelleted, stained with trypan blue (Flow Laboratories, McLean, VA), and counted in a hemocytometer (Improved Neubauer, Reichert Scientific Instruments, Buffalo, NY) (6). In complementary experiments, the viability of HEp-2 cells was determined by using the Live/Dead viability-cytotoxicity assay, according to the manufacturer's instructions (Molecular Probes) (24).

The viability of EPEC strain E2348/69 was determined by incubating the bacterium with each of the three fluidizers under identical conditions used for the adhesion assays. At the end of the 3-h incubation period, serial 10-fold dilutions were placed onto MacConkey agar plates. Results are expressed as the percentage of viable organisms present after fluidization compared with untreated controls.

Statistical Analyses

Results are expressed as means ± SE. Statistical significance was tested by the two-tailed nonpaired Student's t-test (StatWorks, Philadelphia, PA). A P value of <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane Fluidizers Inhibit Adhesion of Bacteria to Host Epithelial Cells

Compared with the control unfluidized cells (Fig. 1A), as determined by the examination of Giemsa-stained slides, the membrane-fluidizing agents hexanol and benzyl alcohol both markedly inhibited EPEC binding to HEp-2 cells at concentrations of 5 and 10 mM, respectively (Fig. 1, B and C). A2C showed a comparable inhibition of bacterial adhesion at a concentration of 2.5 mM (Fig. 1D). Similar inhibition of bacterial adhesion was also seen with each of the other four strains of E. coli when tested with 5 mM hexanol as the membrane-fluidizing agent (Table 1). In contrast, methanol, which does not fluidize the membrane (22), failed to show any detectable inhibition of EPEC adhesion to HEp-2 cells, even at concentrations of up to 50 mM (Fig. 1E). Bright-field microscopy of the treated HEp-2 cells showed some enlargement of the cytoplasm and the formation of pseudopods on the plasma membrane (Fig. 1, B-D) indicative of a more fluid plasma membrane.


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Fig. 1.   Giemsa-stained light microscopy photographs showing adhesion of enteropathogenic Escherichia coli (EPEC) strain E2348/69 to human epithelial cell line HEp-2 cells after 3-h infection in vitro. Compared with unfluidized (A) and methanol-treated cells (E) showing microcolonies of adherent EPEC (arrows), inhibition of bacterial adhesion was observed in cells fluidized with 5 mM hexanol (B), 10 mM benzyl alcohol (C), and 2.5 mM 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) (D). Pseudopodic shape and moderately larger size of HEp-2 cells, indicative of a more fluid plasma membrane, was observed only in treated cells (B, C, and D). Approximate magnification, ×1,000.


                              
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Table 1.   Relative inhibition of Escherichia coli adhesion with hexanol

The three fluidizers showed a dose-dependent inhibitory effect on EPEC strain E2348/69 adhesion to HEp-2 cells, as determined by the quantitative bacterial adhesion assay (Fig. 2). Hexanol at a concentration of 5 mM showed a significant inhibition of adhesion compared with 50 mM methanol after 3 h of infection (86 ± 3% vs. 32 ± 10%) (Fig. 2A). As shown in Fig. 2, B and C, similar results were observed with benzyl alcohol at 10 mM and A2C at 2.5 mM, with 87 ± 12% and 81 ± 9% inhibition of adhesion, respectively. With each of the fluidizers tested, the effect was dose dependent because successively reducing the concentration of the agents led to an abrogation of the inhibitory effect.


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Fig. 2.   Quantitative adherence of EPEC strain E2348/69 to HEp-2 cells after 3-h infection at 37°C. Dose-dependent inhibition of bacterial adhesion to HEp-2 cells was observed with each of the 3 membrane fluidizers. Results are expressed as means ± SE. Each assay was run in duplicate on 3 separate occasions. *A P value of <0.05 was considered significant when effect of 5 mM hexanol compared with 50 mM methanol (MtOH) was evaluated (A).

To determine if the fluidizers affected the viability of HEp-2 cells, trypan blue exclusion and Live-Dead cytotoxicity assays were employed. The viability of HEp-2 cells was not reduced with any of the membrane fluidizers at the maximum concentration used in this study (data not shown). Viable counts of bacteria showed that these fluidizing agents also did not reduce the viability of EPEC strain E2348/69 (Table 2). This showed that the inhibition of EPEC adhesion observed with these agents was not due to the loss of viable bacteria.

                              
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Table 2.   Effect of membrane fluidizers on bacterial viability

Changes in Membrane Fluidity of HEp-2 Cells

The change in membrane fluidity of cultured cells treated with hexanol or benzyl alcohol was measured by assessing fluorescence depolarization of the probe TMA-DPH. To determine the time period over which the steady-state anisotropy parameter remained stable, labeled cells were monitored for a change in anisotropy parameter (Fig. 3). The change in the anisotropy parameter of labeled cells was not significant until 30 min after addition of the probe (n = 6, P > 0.05). As a result, the effect of the fluidizers on changes in the anisotropy parameter was evaluated over the first 20 min after the cells were labeled with TMA-DPH.


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Fig. 3.   Change in steady-state anisotropy parameter of HEp-2 cells labeled with probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Change in anisotropy parameter of labeled but unfluidized cells was not significant (n = 6, P > 0.05) until 30 min after addition of probe (* P < 0.05).

The effects of hexanol (5 mM) and benzyl alcohol (10 mM) on membrane fluidity of HEp-2 cells were then examined. HEp-2 cells fluidized with both hexanol (n = 7, P < 0.005) and benzyl alcohol (n = 4, P < 0.05) showed a drop in the anisotropy parameter at each time point compared with the nonfluidized cells (Fig. 4). This drop in rs (i.e., an increase in membrane fluidity) was observed within 5 min of adding the fluidizers and lasted for the duration of the 20-min assay (data not shown). In contrast, methanol (50 mM) did not significantly reduce the anisotropy parameter of HEp-2 cells compared with the nonfluidized cells (Fig. 4).


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Fig. 4.   Percent change in anisotropy parameter after treatment with TMA-DPH (open bars), TMA-DPH plus 5 mM hexanol (stippled bars), TMA-DPH plus 10 mM benzyl alcohol (hatched bars), and TMA-DPH plus 50 mM methanol (solid bars). Compared with nonfluidized cells, HEp-2 cells fluidized with hexanol (n = 7, Student's t-test, P < 0.005) and benzyl alcohol (n = 4, Student's t-test, * P < 0.05) showed a decrease in anisotropy parameter at each time point. In contrast, methanol did not significantly decrease anisotropy parameter (n = 6, Student's t-test, P > 0.05).

Total fluorescence of fluidized cells did not change over time (Table 3). This finding indicated that the fluorescence lifetime of the probe did not increase over time. Therefore, the observed drop in rs value was due to an increase in the fluidity of the plasma membrane.

                              
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Table 3.   Shift in total fluorescence of fluidized cells over time

Fluidizing Cells in the Early Stages of Infection Is Necessary to Prevent Bacterial Binding

Time course experiments were performed to determine whether the eukaryotic cells need to be fluidized at a particular stage during the course of infection in order to prevent the binding of bacteria. HEp-2 cells were infected with EPEC strain E2348/69, and A2C was then added at 1, 2, or 3 h after bacterial infection. After a 30-min incubation, the Giemsa-staining adhesion assay was performed. Cells treated with A2C within 1 h of infection demonstrated the greatest inhibition of EPEC adhesion (Fig. 5). This effect was completely absent in HEp-2 cells fluidized 3 h after bacterial infection. This result indicated that fluidizing of the eukaryotic cell plasma membrane was effective in inhibiting bacterial adhesion only in the early stages of bacterial attachment.


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Fig. 5.   Photographs of a time-course study showing that an increase in plasma membrane fluidity with A2C was effective in inhibiting adhesion of EPEC strain E2348/69 to HEp-2 cells only during the early stages of bacterial binding. Compared with HEp-2 cells left unfluidized (A), inhibition of adhesion was maximum (B), intermediate (C), and low (D) when A2C was added after infection at 1-, 2-, and 3-h intervals, respectively. Arrows point to microcolonies of adherent bacteria. Approximate magnification, ×1,000.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that increasing the fluidity of the plasma membrane can inhibit bacterial adhesion. By increasing membrane fluidity of host cells through the use of fluidizing agents, we have shown that the binding of the human EPEC strain E2348/69 can be prevented. Two complementary adhesion assays, Giemsa staining and a quantitative adhesion assay (14), were used to demonstrate a dose-dependent inhibition of EPEC adhesion to HEp-2 cells. The specificity of the response was evident because, in contrast to the membrane fluidizers we tested, methanol even up to 50 mM only marginally inhibited bacterial adhesion (Fig. 2). Methanol is a small, single-carbon molecule that is known not to affect membrane lipid fluidity in the concentrations used (22).

Changes in membrane fluidity can alter cellular enzymatic and transport functions, receptor function, and receptor-ligand associations (27, 28). The rationale for employing three different fluidizers in the present study was that the agents are structurally dissimilar. Furthermore, it has been suggested that these agents insert into different regions of the plasma membrane (9, 10, 15, 22). Therefore, a shared nonfluidity-dependent mechanism of action would be unlikely. It is well established that the inner (cytofacial) and the outer (exofacial) hemileaflets of eukaryotic plasma membranes are distinct with respect to their membrane physical properties (9, 10). Differences between the two hemileaflets translate to a varying response of the membranes to agents that perturb their native physical state, including membrane fluidizing agents. For example, in rabbit enterocyte microvillus membranes A2C increases the rates of glucose transport, whereas hexanol reduces the transport of glucose (10, 22). Dudeja et al. (10) showed that A2C preferentially fluidizes the cytofacial leaflet of rat small intestinal brush-border membranes. In contrast, benzyl alcohol preferentially fluidizes the exofacial leaflet of the plasma membranes (10). The precise site of action of hexanol is not known.

The finding in this study that each of the three fluidizers inhibited the binding of EPEC indicates that it is possible to prevent EPEC adhesion regardless of the mechanism of fluidization of the eukaryotic cell plasma membrane employed. This finding also suggests that the receptors for EPEC adhesion are not selectively associated with one hemileaflet of the plasma membrane, perhaps reflecting a transmembrane receptor like that described for the lapine enteropathogenic E. coli strain RDEC-1 (serotype O15:H-) (26). Furthermore, the inhibition of bacterial adhesion appears to be independent of the mechanism of bacterial attachment, because four other E. coli strains, each adhering to the tissue culture cells in a distinct manner, were also prevented from attaching to the fluidized membranes of HEp-2 cells.

Changes in membrane fluidity can affect receptor binding function by changing the displacement of the receptor and by affecting its rotational and lateral motions (27). In addition, alterations in lipid fluidity lead to alterations in the tertiary and quaternary structure of the receptor that could affect the interaction of receptor with bacterial ligands (27). The consequence of these changes in receptor function is a deviation from its native conformation with the resultant inability to correctly recognize and interact with a bacterial ligand.

The inhibitory effects of the fluidizing agents on bacterial adhesion were reversible, because removing the agents before EPEC infection did not influence binding. These results are in agreement with Lustig et al. (19), who showed the reversibility of the effects of A2C on leukemic cells if the cells are washed free of the agent within 2 h of treatment. It was necessary, therefore, to fluidize the cells throughout the process of bacterial adhesion.

To impact binding of prokaryotes, it was essential to increase the membrane fluidity of HEp-2 cells during the early stages of bacterial adhesion (Fig. 5). This finding could indicate that over time the bacterial adhesions cluster eukaryotic receptors with sufficient avidity so as to prevent alterations in the conformation of the receptor in response to membrane fluidization. In addition, the findings suggest that the fluidizer does not alter the interaction of the adherent bacteria; that is, the fluidizers do not behave as antibiotics.

Increases in the membrane fluidity of HEp-2 cells was demonstrated by fluorescence depolarization of TMA-DPH. This technique has considerable advantages over other methods employed to measure membrane dynamics because it offers the sensitivity and simplicity essential for a reliable evaluation of membrane lipid fluidity (28). The probe TMA-DPH possesses a hydrophilic headgroup (25), which ensures that it is aligned parallel to lipid acyl chains and incorporated rapidly into and remains localized within the plasma membrane (16, 17). Nevertheless, the incorporation of any probe into the intracellular membranes is inevitable over time when whole living cells are used. Therefore, it was possible to measure the change in membrane fluidity only over a period of 20 min, since significant intracellular incorporation of the probe occurred after 30 min, in agreement with the findings of Kuhry et al. (16) who showed the penetration of a TMA-DPH label into the intracellular membranes of L929 mouse fibroblasts at the same 30-min time point.

Alterations in rs can be secondary to either alterations in membrane physical properties or a change in the fluorescent lifetime of the probe (22). Unfortunately, the fluorescent lifetime of the probe cannot be directly determined from steady-state measurements. It is directly proportional, however, to the total fluorescence of the probe, a quantity that can be readily determined from the steady-state measurements (22). During the course of measuring the rs parameter in this study, the fluorescence of the probe did not increase (Table 3). This finding indicates that the change in rs was, in fact, secondary to a change in physical properties of the plasma membrane.

It is also possible that the membrane fluidizers affected bacterial membranes. We have not been able to consistently measure changes in prokaryotic membrane fluidity using the steady-state anisotropy method used to measure changes in eukaryotic cell membrane fluidity. Preliminary evidence indicates that when bacteria are incubated in the presence of fluidizers for 3 h, followed by addition to epithelial cells, there is no inhibition of bacterial adhesion. These data provide indirect evidence that the fluidizers employed do not irreversibly affect bacterial membranes. Future studies should evaluate other complementary methods of measuring membrane fluidity, such as pyrene excimer formation by flow cytometry (20), in which the fluidity of prokaryotic and eukaryotic membranes can be determined simultaneously (1).

The clinical significance of this novel mechanism for inhibiting bacterial adhesion requires further study. At least one report has shown that linoleic acid, a fatty acid known to increase membrane fluidity (13), inhibits EPEC adhesion to HEp-2 epithelial cells (5). Although an elucidation of the mechanism of inhibition of E. coli adhesion was not undertaken, it is possible that it was due to an increase in host cell membrane fluidity. Polyunsaturated fatty acids, including linoleic and oleic acids, are known to incorporate into biological membranes and increase membrane fluidity (13). The effects of dietary fatty acids and their derivatives on membrane fluidity need to be examined. Similarly, the effects of detergents, such as bile acids that are normally present in the gut lumen, on membrane fluidity also need to be examined.

It is possible that these in vitro studies could help set the stage for determining the role of altered membrane fluidity in inhibiting bacterial adhesion in vivo. Several studies have shown that feeding animals diets enriched with unsaturated fats can modulate the fatty acid composition and fluidity of plasma membranes (21). For example, Brasitus et al. (4) found that the lipid fluidity of membranes from rats fed fish oil was reduced. Therefore, future work should involve feeding experimental animals diets selected for certain fatty acids so as to change the membrane fluidity, followed by challenge with enteropathogenic bacteria to determine if colonization of the gut is altered. Ultimately, the findings presented herein could form the basis for a novel strategy to prevent adhesion of bacterial pathogens to receptors in the gastrointestinal tract and thereby interrupt the infectious process.


    ACKNOWLEDGEMENTS

We thank Humberto Jijon for assistance with the tissue culture work, and Dr. Zenghua Ma for assistance with the spectrofluorometer.


    FOOTNOTES

Support for this project was provided by grants from the Nutricia Research Foundation, Zoetermeer, The Netherlands; Janeway Child Health Centre, Newfoundland, Canada; and the Medical Research Council of Canada.

A. Ismaili is a recipient of an Ontario Graduate Scholarship. J. B. Meddings is an Alberta Heritage Scholar. P. M. Sherman is the recipient of an A. C. Finkelstein Award from the Medical Research Council of Canada.

This work was presented in part at the Annual General Meeting of the American Society for Microbiology, May 1995, in Washington, DC.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. M. Sherman, Div. of Gastroenterology and Nutrition (Room 8411), The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8 (E-mail: sherman{at}sickkids.on.ca).

Received 29 July 1998; accepted in final form 18 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(1):G201-G208
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