Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

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Ultramicroscopy 106 (2006) 695–702 www.elsevier.com/locate/ultramic

Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM M.A. Beckmanna, S. Venkataramanc,e, M.J. Doktycza,e, J.P. Natarod, C.J. Sullivana, J.L. Morrell-Falveye, D.P. Allisona,b,e,f, a

UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, TN 37996-0840, USA Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA c Department of Electrical and Computer Engineering, The University of Tennessee, Knoxville, TN 37996-0840, USA d Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA e Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123, USA f Molecular Imaging Inc. Tempe, AZ 85282, USA

b

Received 2 December 2005; accepted 10 February 2006

Abstract Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force–distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria. r 2006 Elsevier B.V. All rights reserved. PACS: 018; 022; 024 Keywords: Atomic force microscopy; Enteroaggregative E. coli; Force distance spectroscopy; Bacteria; Immobilization; Live cell imaging

1. Introduction Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen that has been associated with endemic and epidemic diarrheal illness in developing and industrialized countries [1,2]. The pathogenesis of EAEC infection is thought to occur by adherence of the pathogen to the negatively charged intestinal mucosa, most likely of both the small and large intestines, by thin, positively Corresponding author. UT-ORNL Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, TN 37996-0840, USA. E-mail address: [email protected] (D.P. Allison).

0304-3991/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ultramic.2006.02.006

charged, hair-like, adhesive structures called aggregative adherence fimbriae (AAF). A pathological process ensues and is initiated by one or more enterotoxins [2,3]. In Gram-negative bacteria, such as E. coli, the cell wall is positioned outside the cell membrane and contacts the environment. This structure consists of an outer lipopolysaccharide (LPS) membrane overlying a gel-like periplasm and a thin peptidoglycan inner layer [4,5]. Approximately 75% of the outer membrane of the cell wall is LPS while the remaining 25% is composed of membrane proteins that may be compactly folded and form dense structures that probably represent the rigid regions of the cell wall [6,7]. The outer cell wall may also contain a variety of external

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structures, such as fibrils, fimbriae, pili, and flagella [8,9]. The combination of all of these structures determines the physicochemical cell surface properties of a particular bacterial strain. Working with the pathogenic E. coli strain EAEC-042, Nataro’s laboratory discovered a novel protein they named ‘‘dispersin’’ that is thought to play a significant role in the pathogenic sequence. This 10-kDa low molecular weight protein is secreted to the exterior environment of the bacteria and remains non-covalently associated with the cell surface. The positively charged dispersin is believed to cover the negatively charged LPS of the cell wall. Either the repulsion between dispersin and the positively charged AAF fimbriae or the neutralization of the LPS layer by dispersin effectively allows the fimbriae to move away from the cell surface. The extended fimbriae can then make contact with the intestinal mucosa and initiate colony formation. Dispersin is also thought to contribute to pathogenesis by allowing individual progeny of established colonies to swim freely through the mucosa to establish new foci of infection. A mucus penetration assay revealed that a dispersin deletion mutant (042aap) was more than an order of magnitude less efficient at penetrating the mucin column than was the wild type parent strain [2,3]. The design of diagnostic and treatment protocols for this pathogen is dependent upon knowledge of the functions of the fimbriae the dispersin protein and the surface properties they impart on the cell wall of EAEC in vivo. Historically, it has been recognized that important aspects of microbial behavior are controlled by the physicochemical properties of the cell wall [8–14]. Gaining an understanding of the elastic properties of cell surfaces is important since the degree of elasticity will depend on the composition of the various structural components [4,7,11,15–17]. Measuring the elastic properties of living cells qualitatively has been demonstrated by Hoh and Schoenenberger using an AFM as a force sensor [18–20]. The tip of an AFM can be used to indent soft samples, and the force versus indentation measurement provides information about the viscoelasticity [18,19,21]. Simultaneously, data in the form of load force (V) versus displacement distance (nm) are recorded to plot a force–distance (FD) curve. In principle, this gives a plot of the force required to achieve a certain depth of indentation (deformation) from which viscoelastic parameters can be determined [22–24]. In this paper, wild type and the genetically modified dispersin mutant of the EAEC 042 strain are examined to determine if changes of surface structure mediated by dispersin can be registered by means of AFM spectroscopy. 2. Materials and methods 2.1. Preparation of mica surfaces for atomic force microscopy A hole punch (Ralmikes TOOL-A-RAMA, South Plainfield, NJ) was used to punch 3/4 in diameter mica

disks out of a flat mica sheet. The disks were freshly cleaved on both sides by removing the outer surface with scotch tape. Gelatin solution was prepared by dissolving 0.5 g of gelatin (Sigma G-6144) and 10 mg of chromium potassium sulfate in 100 ml nanopure deionized water at 90 1C. After cooling to 60 1C, the mica disks were vertically dipped into the solution and allowed to air-dry overnight supported on edge on a paper towel at a steep angle [25].

2.2. Preparation of bacteria for atomic force microscopy Bacteria used in this study were EAEC 042pet, essentially the wild-type bacterium, but carrying a mutation in the Pet toxin for safe laboratory use and EAEC 042aap a dispersin mutant of EAEC 042. Bacteria were maintained on Luria broth (LB) agar plates and these cultures were used to inoculate both LB broth cultures and LB agar plates. Bacteria grown on agar plates were freshly inoculated and incubated at 37 1C for 5 h so that cells were in logarithmic phase. The cells were prepared for imaging by scraping a small quantity of the bacteria off the culture plate with a sterile loop and transferring the cells into a micro-centrifuge tube containing 0.6 ml of distilled water. After mixing, the sample was centrifuged at 10,000 rpm (9.3 rcf) for 10 min. The wet pellet was suspended in 0.6 ml of distilled water and 100 ml was pipetted onto the gelatin-treated mica disk, allowed to stand for 10 min, rinsed vigorously in a stream of deionized water, and either allowed to dry for imaging in air or covered with distilled water, in a wet cell, for liquid AFM imaging. Logarithmic phase bacteria cultures, grown in LB broth, were prepared similarly starting with 0.6 ml of broth culture and following the procedure described for plated bacteria.

2.3. Atomic force microscopy imaging Liquid (Fig. 1) and air (Fig. 2) dried samples of both the wild type (042pet) and the dispersion mutant (042aap) were imaged in a PicoPlus AFM (Molecular Imaging Inc., Tempe, AZ). Imaging in liquid was done with the AFM operating in MacMode with a scan rate of 1.0 line/s with 512 points per line using silicon cantilevers with a manufactures stated spring constant of 2.8 N/m. Topographic and amplitude images were recorded and the only processing done was first order flattening. Air-dried samples were placed in an environmental chamber for imaging where the relative humidity was maintained at 12% by flooding the chamber with dry nitrogen. Imaging was done in contact mode with 512 points per scan line collected at a scan rate of 1.5 lines/s using the same silicon cantilever. Topographic and deflection images were recorded and the only processing done was first-order flattening.

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Fig. 1. Topographic MacMode AFM images of the wild type pathogenic E. coli strain EAEC 042pet grown on LB agar (a) and in LB broth (c) and the non-pathogenic dispersin mutant EAEC 042aap grown on LB agar (b) and in LB broth (d) are shown imaged in water. Line scans (e–h) taken through their corresponding images (a–d) show an average height of 600–800 nm. Logarithmic cultures were used as evidenced by the presence of dividing cells in all the images.

2.4. Force–distance spectroscopy All force–distance curves were collected on samples in deionized water. Each experiment consisted of collecting

force curves on four different samples; 042pet grown on agar, 042pet grown on broth, 042aap grown on agar and 042aap grown on broth. A single silicon nitride cantilever with a manufactures suggested spring constant of 0.1 N/m

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Fig. 2. The samples used in Fig. 1, were allowed to dry and imaged in contact mode in an environmental chamber, purged with dry nitrogen, at a relative humidity of 12%. Line scans (f, g) through the topographic image (a) of 042pet grown on agar shows a height of 200–300 nm due to dehydration of the sample. Deflection mode images of 042pet grown on LB agar (b) and in LB broth (d) and 042aap grown on LB agar (c) and in LB broth (e) show a marked dehydration of the bacteria without lysis. Both 042pet and 042aap are more resistant to dehydration when grown on agar.

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was used in each experiment and once the laser spot was adjusted on the backside of the cantilever, for the maximum signal in the photosensitive detector, it was not moved during the course of an experiment. In each experiment force curves on bare mica were collected in deionized water at the beginning end and sometimes in between sample measurements to serve as a reference. In every experiment approximately 50 force curves were collected on both bacteria and the gelatin coated mica surface. All force curves were collected with a sweep duration of 1.0 s with 2000 points/curve. 2.5. Analysis of force curves We used the gradient analysis method described by Li and Logan to analyze the approach curves presented in this paper [26]. This methodology divides the force curve into four separate regions as shown in Fig. 3: (A) a region where the cantilever tip is approaching the sample, but has not made contact, (B) a region where there is interaction with the surface without contact, (C) a region where initial contact is made with the surface, (D) a region where hard contact is made with the surface. A semi-automatic user interface algorithm was developed to analyze force curves. An average of 50 curves was plotted and the four regions mentioned above were defined by user input. Instantaneous slopes at all the data points are plotted on a secondary Y-axis. This is done by determining the slope between consecutive points in the approach curve. This allows for accurate identification of changes in slope that are hard to determine accurately by simply looking at the force curve. Points are selected to divide the curve into 4 different regions (A–D) by observing the changes in slopes at these points. Once the regions are separated, spring constants for a particular slope can be calculated. For example, a line is drawn that passes through the most linear section of the curve in region D and the slope of this line is calculated (m1). A second line corresponding to the small linear region within C is drawn and the slope for the same is also calculated (m2). Higher measured value of the slope denotes a more rigid surface. The products of these slopes multiplied by the spring constant of the cantilever (0.1 nN/ nm) are calculated to give a spring constant in nN of the measured surface. The algorithm was implemented using MATLAB 7.0. 3. Results and discussion AFM imaging of cells in liquid was accomplished using MacMode, an intermittent contact mode, where the cantilever, with a magnetic coating on its backside, is induced to oscillate at its resonant frequency by application of a magnetic field [27,28]. This mode of operation allows for the least amount of force to be applied to the sample by the AFM tip. In Fig. 1 topographic images of all four of the bacterial sample preparations are shown imaged in

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water. Care was taken to rapidly image the bacterial samples after they were removed from their respective growth media, centrifuged to a pellet, and suspended in water. The EAEC 042pet wild type strain is shown grown in agar (a) and broth (c) while the non-pathogenic dispersin mutant 042aap is shown grown in agar (b) and broth (d), respectively. In all cases, the cultures contained dividing cells, indicating that the cells were harvested in logarithmic phase. In these images, the wild type and mutant cells appear to have a very similar morphology. This is expected because the depth of field of the topographic image does not allow for sensitive imaging of both the entire height of the bacteria and fine structure. For example, pili, or flagella that might be present on the mounting surface would be difficult to see in the topographic image. A line scan arbitrarily drawn through the images shows a reasonably consistent height 600–800 nm for all the bacterial preparations. A line scan that passes perpendicular through bacteria shows an exaggerated width of roughly 2 mm. This is due primarily to the tip being shaped as a pyramid such that a convolution of the image results [29]. The samples that were imaged in air were the same samples we imaged in liquid. After drying, images were taken in contact mode in a humidity-controlled environment. These AFM images of both the 042pet wild type and 042aap dispersin mutant are presented in Fig. 2. One effect of drying can be clearly seen in the topographic image (a) of 042pet grown in agar. In this image the cross section height of the bacteria shown in both the line scans (f,g) is roughly 200–300 nm as opposed to the height measured in liquid of 600–800 nm. Similar effects of drying were seen for the other preparations. This apparent difference is due to dehydration and not lysis since intracellular material such as DNA and proteins are not present on the surface. What this indicates is that the bacterial cell wall lacks sufficient rigidity to maintain its form when water is removed. The images shown in Fig. 2b–e are deflection images where true height values are sacrificed to allow for a more sensitive view of surface features. We find that when cells are isolated they tend to maintain their rod like shape while cells in close contact show a more pronounced collapse of the cell wall. This is clearly seen in (b) where the isolated cells of 042pet grown in agar are more normal looking as opposed to (c) where 042aap grown in agar shows a normal-looking cell on the periphery but the closely packed cells definitely show evidence of severe dehydration. One observation that we have made in comparing images of all different preparations is that cells grown on agar appear to maintain their characteristic rodshaped appearance better than cells grown in broth. This can also be seen in the images where 042pet (b) and 042aap (c) grown in agar appear to be less dehydrated than (d) 042pet and (e) 042aap grown in broth. It is also interesting to note that 042pet has what appear to be flagella when grown on both agar and broth, although more flagella are observed when grown on agar, while 042aap does not exhibit any flagella.

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Gel

m1

m1 = 0.2200 m2 = 0.1040

-0.2

0

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-700

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0 Slope

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B 43 nm

C 34 n m

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Slope

0

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5 m2

m1 = 0.2110 m2 = 0.0800

-0.2

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-0.4 -5 -1800 -1750 -1700 -1650 -1600 -1550 -1500 -1450 -1400 Piezo movement (nm)

Piezo movement (nm) 042 PET - broth

042 AAP - broth

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m2 m1 = 0.0690 m2 = 0.0400

-0.1

-0.2 -1900

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-1700

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m2

m1 = 0.0780 m2 = 0.0450

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Piezo movement (nm) 042 PET - agar D C B

D

29 30 nmnm

0

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m2

5

m1 = 0.1330 m2 = 0.0600

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0

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042 PET - agar

A

Approach curve Slope

-5 -1800 -1700 -1600

-5 -1500 -1400 -1300 -1200

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C B 30 35 nm nm

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m2

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C B 32 66 nm nm

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C B 34 34 nm nm

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10

Deflection (nm)

B + C m1 = n m

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m1 = 0.0810 m2 = 0.0576

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150 200

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700

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Fig. 3. Force curves of the approach and contact of the AFM cantilever with the surface of wild-type EAEC 042pet and the dispersion mutant EAEC 042aap bacteria are presented. The force curves were subjected to gradient analysis so that four areas of interest (A) approach to the sample surface, (B) interaction with the sample without contact, (C) initial contact with the sample, and (D) hard contact with the sample, could be identified. For wild-type 042pet grown on agar hard contact with the cell surface (m1 ¼ 0.133) shows a rigid surface but loses most of this rigidity (m1 ¼ 0.069) is lost when grown on broth. For the 042aap dispersin mutant there is very little difference in rigidity when grown on agar (m1 ¼ 0.081) and when grown on broth (m1 ¼ 0.78). Values for m2 where initial contact with the bacteria is made are nearly identical 042pet (m2 ¼ 0.06) and 042aap (m2 ¼ 0.056) when grown in agar. The values of m2 for bacteria grown on broth are also nearly identical for 042pet (m2 ¼ 0.040) and 042aap (m2 ¼ 0.045), but are markedly lower than the values on agar. These values can be converted to spring constants by multiplying m1 or m2 values by the cantilever spring constant.

In addition to imaging, the sensitivity of the AFM cantilever can be exploited to measure forces on the order needed to rupture a single hydrogen bond [30]. Forces

between and within biomolecules have been measured [31], and most importantly for this application, force measurements have also been made on sample surfaces, including

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bacterial cell surfaces [5,14,32]. Force measurements on sample surfaces are usually made in contact mode by approaching the sample surface with the cantilever tip, contacting the surface with the cantilever tip, and withdrawing the cantilever. In this paper, we have concentrated only on the approach and contact of the cantilever tip with the bacterial cell surface and have not included the withdrawal of the tip from the surface. The result of one set of the experiments is presented in Fig. 3. In this study we repeated the experiment three times and the results were in agreement. In all of our experiments force measurements were made with the sample in water with a mica surface used as a control. Force measurements were made on mica before, midway and at the end of the experiment to determine if there was any change in the spring constant or laser position as evidenced by a change in slope. For example, in the data presented in Fig. 3, the slope (m1) for mica was 0.223, 0.235, and 0.217 nm, respectively for the beginning, midpoint, and end of the experiment. This would indicate that nothing happened during the course of the experiment to change the spring constant of the cantilever. In all experiments the same cantilever was used in order to eliminate the problem of variation in the spring constant of different cantilevers affecting the measured values of slopes. Also, once the laser spot was adjusted on the backside of the cantilever, for maximum signal in the position sensitive diode, no further adjustment was made during an experiment. In Fig. 3, section (A) where the cantilever is approaching the surface, the approach curve is linear and the calculated gradient slope stays at zero. The movement of the cantilever as it approaches the surface and the tip contacts the sample, can be accurately followed by the piezo movement in nm. For mica surfaces the region of interaction with the surface without contact (B) and the region where initial contact with the surface is made (C) are hard to differentiate from the approach curve. However, the gradient analysis detects the difference in slope in this region and in other regions and is used to define areas (A–D). Although (B) and (C) regions are not differentiated on the mica surface there is a distance of 8 nm that the cantilever moves before hard contact with the surface (D) can be identified. In the other graphs that show the results on gelatin-coated mica and the four bacterial samples, (B) and (C) areas are well differentiated. In area (B) where the tip is being repulsed and the cantilever is starting to bend in a nonlinear way the tip is not actually contacting the surface, but is likely being repulsed by electrostatic forces or by structures external to the cell wall such as fimbriae, pili, and flagella [8,9,26]. Again differentiating between (B) and (C) areas from the approach curve is difficult but they show up as changes in the calculated slope. The approach curve in area (D) is most often used to calculate the spring constant of a surface. In order for this to be accurate it is necessary to measure the spring constant of the cantilever [33,34]. However, in this study we have not calibrated the cantilever spring constant since we are more interested in

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comparing results between the wild type EAEC 042pet strain that produces dispersin and is pathogenic with the EAEC 042aap dispersin mutant that is not pathogenic. Looking at the slope of m1 for all the surfaces we would conclude that the mica and gelatin surfaces are significantly more rigid than the bacterial samples. Bacteria grown on agar are more rigid than bacteria grown in broth. This is also evident when comparing images collected in air (Fig. 2) where the agar-grown cells appear less dehydrated than do broth grown cells. The wild type 042pet grown on agar has the most rigid (m1 ¼ 0.133) cell surface, but loses most of this rigidity (m1 ¼ 0.069) when grown in broth. The 042aap dispersin mutant is somewhat more rigid (m1 ¼ 0.081) when grown on agar but does not appear to lose much rigidity (m1 ¼ 0.78) when grown on broth. The values that we obtain for m2, where initial contact with the bacteria is made are virtually equal for 042pet (m2 ¼ 0.06) and 042aap (m2 ¼ 0.056) grown in agar. These values are also equal for 042pet (m2 ¼ 0.040) and 042aap (m2 ¼ 0.045) grown in broth. However, the m2 values show that initial contact of the cantilever tip with the surface, in both the wild type and dispersin mutant, shows greater rigidity in bacteria grown in agar as opposed to broth grown bacteria.

4. Conclusion We have evaluated AFM images and evaluated force measurements on a pathogenic strain of enteroaggregative Escherichi coli (EAEC) that produces a severe diarrhea in humans. The wild type strain 042pet produces a protein, dispersin, that is implicated in pathogenesis. In this paper a mutant of 042pet that does not produce dispersin, 042aap, is compared to the wild type to determine if physical differences might be present that could also be implicated in the pathology of this bacterial strain. We have found some differences in cell rigidity between the wild type and mutant strains. Further we have demonstrated that the gelatin-based bacterial cell mounting procedure is compatible with the collection of elasticity measurements and that measurements collected in air can be compromised by drying of the bacteria. A deeper understanding of cell rigidity will be the subject of future research efforts.

Acknowledgments This research was funded by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), US DOE Office of Biological and Environmental Sciences Medical Sciences Division, NIH Grant 1R41GM071143-01 to DPA and by NIH Grants R01 AI-33096 to JPN. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US Department of Energy under Contract No. DE-AC0500OR22725.

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