Atomic force microscopy evaluation of the effects of a novel antimicrobial multimeric peptide on Pseudomonas aeruginosa

Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198 – 207 www.nanomedjournal.com

Experimental

Atomic force microscopy evaluation of the effects of a novel antimicrobial multimeric peptide on Pseudomonas aeruginosa Greta Rossetto, MS,a Paolo Bergese, PhD, a Paolo Colombi, PhD,a,⁎ Laura E. Depero, a Andrea Giuliani, PhD,b Silvia F. Nicoletto, MS,b Giovanna Pirri, MD, MS b a

INSTM and Laboratorio di Chimica per le Tecnologie, Università di Brescia, Brescia, Italy b SpiderBiotech S.r.l., Colleretto Giacosa, Torino, Italy Received 27 April 2007; accepted 22 June 2007

Abstract

In this article we evaluated by atomic force microscopy (AFM) the effects of the (novel) tetrabranched antimicrobial peptide SB006 on morphology and mechanical properties of the gramnegative bacterium Pseudomonas aeruginosa. AFM imaging showed that SB006 causes the appearance of significant fragmentariness in the bacterial membrane and a severe volume decrease. Quantitative evaluation of the degree of fragmentariness was allowed by a new ad hoc image analysis procedure. The rigidity of the treated and untreated bacteria was measured through AFM tip nanoindentation measurements, and no differences registered. These results support the membrane interaction hypothesis, according to which SB006 targets the bacterial membranes and disrupts their permeability (allowing the leakage of cytoplasmic material and the subsequent shrinkage), but it does not affect the bacterium wall, which determines its rigidity. © 2007 Elsevier Inc. All rights reserved.

Key words:

AFM; AFM tip nanoindentation; Antimicrobial multimeric peptides; Bacteria

The number of bacterial strains that are resistant to ordinary antimicrobial agents is increasing [1]. Antimicrobial peptides are a new family of antibiotics that have stimulated research and clinical interest [2-4] as a solution to this emerging problem. Most antibacterial peptides are components of the innate immunity of animals and plants against microbial infections [5,6]. Cationic antimicrobial peptides are generally defined as peptides of less than 100 amino acid residues [7,8] with an overall positive charge, imparted by the presence of multiple lysine and arginine residues, and a substantial portion (30% or more) of hydrophobic residues [9,10]. These peptides can possess antimicrobial activity against gram-positive and gramThe authors declare competing financial interests. A. Giuliani is an executive board member and minor shareholder of SpiderBiotech which is developing antimicrobial peptides. G. Pirri is head of research and minor shareholder of SpiderBiotech. ⁎ Corresponding author. INSTM and Laboratorio di Chimica per le Tecnologie, Università di Brescia, Via Branze 38, 25123 Brescia, Italy. E-mail address: [email protected] (P. Colombi). 1549-9634/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2007.06.002

negative bacteria, fungi [11], and protozoa [12], and have demonstrated minimal inhibitory concentrations (MICs) as low as 0.25-4 μg/mL [13]. Because of their low resistance to plasma and serum proteolytic activity [14,15], generally natural cationic peptides show high in vitro activity and a limited in vivo activity. Therefore, it is necessary to resort to different strategies to increase peptide stability for therapeutic application. Particularly, multimeric peptides stand out for their remarkably increased in vivo half-life and for their enhanced antimicrobial activity with respect to their linear homologues [15,16]. The cationic charge of these peptides ensures electrostatic affinity with the negatively charged bacterial outer membrane, whereas the hydrophobic portion of the peptide guarantees interaction with the membrane lipid double layer and facilitates the peptide entry into the cell. In general, cationic peptides show lower selectivity for eukaryotic cells essentially because their membranes have no net charge, whereas the presence of cholesterol reduces cationic peptides

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

activity because of either stabilization of the lipid bilayer or interactions between cholesterol and the peptide. Despite their importance, antimicrobial peptide mechanisms of action are still not fully understood, and a broad variety of modes of action has been proposed. The mechanism of action on gram-negative organisms, which is the best studied, involves the initial displacement of Mg 2+ and Ca 2+ cations that stabilize lipopolysaccharides [17]. This mediates the formation of perturbed areas, through which the peptide translocates the outer membrane by a process termed self-promoted uptake [1,18,19]. The peptide then associates with the outer monolayer of the cytoplasmic membrane. It is at this point that membranedisruptive and non-membrane-disruptive mechanisms diverge, depending on whether this reorientation leads to perturbation of the integrity of the cytoplasmic membrane or to peptide translocation into the cytoplasm, targeting other cell components such as cytoplasmic anionic molecules like DNA or enzymes [1,13,19-21]. Atomic force microscopy (AFM) has potential as an effective tool to gain better understanding of peptides’ mode of action on the bacterial membrane, because it allows both nanometer-scale quantitative imaging and evaluation of mechanical properties [22-24]. Even though AFM may be performed in liquid, image resolution is not satisfactory because of the noise due to the poor adhesion of bacteria to the substrate and the motility of their floating appendices [25,26]. Therefore, even though the extent of surface alteration induced by dehydration is not well established [25-29], several authors preferred air imaging to evaluate bacteria changes due to antibiotic effects, because this operation environment guarantees significantly higher resolution. This approach is also accompanied by remarkable ease of sample preparation and handling [30], and by a stable adhesion of the cells to the substrate. AFM morphology studies on the effects of antimicrobial peptide PGLa on Escherichia coli conducted in both physiological solution and air have been reported by da Silva et al [31]: peptide-altered cells in liquid environment lost their adhesion to the substrate and were removed as a result of the contact of the tip during the scanning, whereas imaging in dried conditions showed a substantial increase in the top surface roughness. Only a few other articles regarding AFM studies on the effects of membrane-active compounds on bacteria are present in the literature, and these studies were conducted in air. Kasas et al [30] studied the action of penicillin on Bacillus subtilis, finding dramatic changes in morphology. Braga and Ricci [32] studied the effects induced by β-lactam antibiotics on Escherichia coli and observed different forms of cell damage ranging from formation of holes in the outer membrane to complete lysis of the cell as shown by flattening in the bacterial morphology. Sharma et al [33] observed flattening of the middle region and bulging at apical ends of E. coli exposed to RISUG (a contraceptive drug possessing antimicrobial properties), whereas Peng

+

199

et al [34] examined La effects on E. coli and evidenced an increase in root-mean-square roughness of treated bacteria envelopes. Meincken et al [35] evaluated the effects on E. coli by three different natural antimicrobial peptides observing distinct changes in cell envelope morphology as a consequence of peptide action: formation of grooves and porelike lesions as well as collapse of the cell structure at the apical ends for melittin, formation of small (~50 nm) outer membrane protovesicles for magainin, and an intermediate behavior for antimicrobial peptide PGLa. Li et al [36] examined morphostructural damage on E. coli and P. aeruginosa cells induced by different concentrations of Sushi peptide S3. They observed the appearing of indentations on the surface of some cells as well as some micelle-like structures arising from lowconcentration treatment and the leakage of a large amount of fluid from the partially disintegrated cell in the case of higher S3 concentration. AFM also allows investigators to probe the mechanical behavior of biological samples. Mechanical analysis is performed through AFM-tip nanoindentation experiments, which consist of monitoring the force acting on the cantilever while it is brought in contact with the sample and retracted. The output of an AFM-tip nanoindentation experiment is the plot of the force registered by the cantilever versus the scanner z-displacement. Hereafter we will refer to it as force-distance curve. Nanoindentation measurements on bacteria have been carried out in other studies [31-37], which have assessed different properties according to the model used to analyze the cell behavior under loading condition. This article focuses on AFM investigation of the antimicrobial multimeric peptide SB006 membrane action on the gram-negative strain P. aeruginosa (American Type Culture Collection [ATCC] No. 27853). The original sequence of this peptide was obtained by selecting a large 10mer phage peptide library against whole E. coli cells. The peptide SB006 (previously referred to as M6) was selected after optimization of the original sequence and synthesized as described by Pini et al [15] as the tetra-branched multimeric form of the linear sequence QKKIRVRLSA. SB006 showed enhanced stability to natural degradation and improved bactericidal activity against a large panel of gramnegative bacteria. In particular, the MICs of the peptide against P. aeruginosa ATCC 27853 was assessed to be 4 μg/ mL. In time-kill experiments it showed rapid bactericidal activity against P. aeruginosa, reducing an inoculum larger than 10 7 colony-forming units (CFU) by N99.9% in 4 hours at a concentration of 16 μg/mL. Membrane permeabilization was evaluated through the β-galactosidase release test, which showed that permeabilization rate depends on peptide concentration and that at 16 μg/mL SB006 is able to permeabilize bacteria in less than 1 minute [15]. AFM imaging operation and AFM-tip nanoindentation experiments allowed us to assess and integrate these preliminary results.

200

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

Materials and methods Peptide synthesis The peptide was synthesized on (9-fluorenylmethoxy carbonyl)4Lys2-Lys-β-Ala Wang resin. Side-chain-protecting groups were trityl for glutamine, tert-butoxycarbonyl (Boc) for lysine,2,2,4,6,7-pentamethyldihydrobenzofuran-5sulfonyl (Pbf) for arginine, and tert-butyl ether (tBu) for serine. Peptides were then cleaved from the resin and deprotected by treatment with trifluoroacetic acid, containing water and triisopropylsilane (at a 95:2.5:2.5 ratio). Crude peptides were purified by reversed-phase chromatography with a Phenomenex C12 Jupiter Proteo column. Identity and purity of final products were confirmed by MALDI-TOF (matrix-assisted laser desorption ionization–time-of-flight) analysis (Brucker, AXS Inc., Madison, WI). Sample preparation Bacterial culture was grown for 18 hours at 37°C with vigorous shaking in a 50-mL Falcon test tube with 5 mL Mueller-Hinton broth. The incubation period led to a concentration of ~10 9 CFU/mL. After incubation, 1 mL of culture broth was first centrifuged for 3-4 minutes at 1000g. The bacterial suspension was then washed in phosphatebuffered saline (PBS) and centrifuged for a second time and finally suspended in 1 mL of PBS. A sample of 10 μL of the bacterial suspension (∼10 7 CFU) was incubated for 30 minutes at 37°C with an equal volume of PBS peptide solution at 2× concentration, obtaining the following final concentrations of SB006: 4 μg/mL (MIC), 8 μg/mL, and 64 μg/mL. After the incubation, a sample of 5 μL was applied to fresh-cleaved mica and air-dried. Samples prepared in such a way remain stable for weeks [38]. AFM experiments AFM experiments were carried out using a JEOL (Tokyo, Japan) scanning probe microscope equipped with a widearea scanner (maximum x-y movement = 50 μm; maximum z-movement = 8 μm) and a four-segment photodetector for cantilever deflection monitoring. Rectangular silicon cantilevers NSG10 (NT-MDT, Moscow, Russia) for noncontact operation were used. For NSG10 cantilevers, the nominal spring constant ranges between 5.5 N/m and 22.5 N/m, with the typical value being 11.5 N/m. We evaluated the actual spring constants of the cantilevers used by resonant frequency analysis, and we obtained spring constants ranging between 8.13 N/m and 31.90 N/m. The cantilever characteristic dimensions were measured by scanning electron microscopy (SEM). Bacteria images were collected in noncontact amplitude modulation mode (contact mode was discarded because it led to bacteria deformation). Multiple AFM-tip nanoindentation experiments were performed on each sample by the same cantilevers used for

imaging. Scanner displacements were limited to 70-100 nm, and bacteria were indented in different positions so as to assess the variability of mechanical properties along the bacterial cell surface. To check possible permanent deformations due to tip-sample interaction, indented bacteria were imaged before and after each nanoindentation experiment. Image analysis and force-distance curve analysis AFM imaging operation allows us to acquire quantitative nanometer-scale topographical information of the observed object. It stores data relating to the absolute height in matrix form and visualizes them in a 512 × 512 pixels image. Highmagnification images (maximum image size 3 × 3 μm 2) were analyzed to evaluate the average height of bacteria and the degree of fragmentariness of bacterial envelopes. Average heights were evaluated to obtain quantification of bacterial volumes. The working equation was applied both on the bacterium surface and on background: X h ¼ 1=A hðiÞ ð1Þ where A is the selected area expressed in number of pixels and h(i) is the absolute height of the i-pixel included in A. The bacterial average height, H, was then calculated as the difference of the bacterial absolute average height (hbact) and the background absolute average height (hbg) (H = hbact – hbg) (see Figure 1 and the Results and Discussion). For each concentration at least 10 distinct P. aeruginosa cells have been analyzed so as to obtain statistical relevance. Because envelope fragmentation was the most evident effect of SB006 treatment, we regarded as necessary its quantitative evaluation. In previous studies [31,33,34], surface fragmentariness induced by chemical treatment was evaluated in terms of roughness, comparing the root mean square of native and treated bacteria envelopes. Meincken et al instead studied the surface corrugation effects of lytic peptide by calculating the surface roughness as the arithmetic average of the surface height deviations from the average plane [35]. Because both of these methods are appropriate only for zero-averaged or periodic geometries, we realized they are not suited for the evaluation of the extensive damage to the whole bacterial cell that we observed as a consequence of SB006 peptide treatment. Thus, we developed an alternative method to evaluate envelope irregularity. We selected, within each image representing a bacterium, only the pixels included between the topmost bacterium value and 80% of it. In this way individual islands emerge from the bacterium image. The number of islands (with the caution of omitting islands smaller than 16 pixels) was taken as the parameter P characterizing the bacterium envelope irregularity (see Figure 2 and the Results and Discussion section). To the best of our knowledge this is the first time that bacterium envelope irregularity has been evaluated in this way. Concerning AFM-tip nanoindentation experiments, we gained information on stiffness and deformability of bacteria exploiting the approach branch of various force-distance

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

201

Fig 1. Consecutive steps of the image analysis process performed for the evaluation of the average height of bacteria. A, Raw AFM image of the bacterium. B, Determination of the absolute average height of the bacterium, hbact1, which is evaluated on the bacterium adhesion area that is displayed in blue. C, Determination of the absolute average height of the background, hbg, evaluated analogously to hbact1. Accordingly, the average height of this bacterium, H, results in H = hbact1 – hbg = 205 nm.

curves [39] acquired for each peptide concentration. Because of sample softness and deformability, force-distance curves registered on a soft samples show a lower slope respect to such curves registered on a rigid substrate, and it is actually

this slope reduction that conveys information regarding material mechanical properties (see Figure 3). The approach branch of each force-distance curve acquired on bacteria was interpreted by modeling the cantilever-bacterium system as

Fig 2. Evaluation of the bacterium envelope degree of fragmentariness by means of the parameter P. A, Raw (A.1) and processed (A.2) AFM images of an untreated bacterium. B, Raw (B.1) and processed (B.2) AFM images of a bacterium treated with SB006 at the concentration of 64 μg/mL. Between each raw image and the corresponding processed one we reported the brightness z-scale. Panels A.2 and B.2 show, in white, the islands formed by those pixels whose absolute height is included between the topmost bacterium value and 80% of it. The number of white areas displayed in the processed image is taken as the parameter P representing the bacterium envelope irregularity. In this case, the untreated bacterium (A) is characterized by P = 2, whereas the bacterium treated with SB006 at a concentration of 64 μg/mL (B) is characterized by P = 5.

202

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

Fig 3. Ideal force curves obtained on a rigid (A) and on a soft (B) sample. At 1.A and 1.B the cantilever tip is far away from the sample, and there is no interaction between the two. At 2.A and 2.B the tip and the sample are brought into contact. On the soft sample the resulting load causes an elastic deformation of the sample, which does not occur in the case of the rigid substrate. As a consequence, for the soft sample the same z-displacement gives a lower applied force than for the case of the rigid sample. At 3.A and 3.B the tip is withdrawn from the sample. For the rigid sample the approach and retraction branch of the force-distance curve overlap. For the soft sample, because of viscoelastic properties or permanent deformation of the sample, the force-distance curve instead shows hysteresis. At 4.A and 4.B the tip adheres to the sample. At 5.A and 5.B, when the force reaches the maximum value (adhesion force), the tip “jumps off contact” from the sample.

two springs connected in series. The equivalent spring constant of each indented bacterium was evaluated for every nanoindentation measurement by the formula [40]: kbact ¼ ðkN sÞ=ð1  sÞ

ð2Þ

where kN is the cantilever spring constant, s = Δd/Δz, d is the cantilever deflection (according to the equation = kNd), and z is the scanner displacement. Even though the parameter kbact does not provide specific information on the bacterium surface structure, nevertheless it is effective in conveying information of the bacterium as a whole. For every nanoindentation measurement we also evaluated the adhesion force between the cantilever tip and the bacterium as the

maximum attractive force arising from the interaction when they are brought apart (Figure 3). Scanning Electron Microscopy (SEM) SEM observations were carried out with an Evo 40 microscope by Leo (now Nanotechnology System Division of Carl Zeiss, SMT, Cambridge, UK). The samples, being nonconductive, were coated with gold (few nanometers) and imaged with secondary electrons in vacuum conditions. Results and discussion Scanning in noncontact mode gave nanometer-scale resolution AFM images (we could recognize polar flagella

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

203

Fig 4. AFM images at different magnifications in which it is possible to recognize bacterial polar flagella. To highlight the flagella, the brightness of each squared area was adjusted. Because 10 nm is the typical height of a cross-sectional profile of the observed flagellum, in the squares every height above 10 nm is visualized with the maximum brightness. A and B correspond to the sample treated with SB006 at a concentration equal to 8 μg/mL, whereas C corresponds to the sample treated with an SB006 concentration equal to 64 μg/mL.

in several bacterial images; Figure 4). Bacterial adhesion to mica was successful, in agreement with previous reports [25,26,30,33,34]. Various bacterial surface densities per unit area were tested, and a surface concentration of ~10 7 cells/

cm 2 proved to be desirable for easy individuation of bacteria with AFM. Moreover, such a surface concentration allows to overcome the difficulties caused by the presence of PBS residuals, consisting in salt crystals that are several microns

Fig 5. AFM images of bacteria at different concentrations of SB006, which show visible morphological alterations due to SB006 treatment. A, Untreated bacterium, characterized by a smooth and close-knit envelope. B, Bacterium treated with SB006 at a concentration equal to the MIC (4 μg/mL), showing initial morphological damage. It is evident that the bacterium starts to collapse and is crossed by a longitudinal rift. C and D, Representative bacteria treated with SB006 at concentrations above the MIC (respectively, 8 μg/mL and 64 μg/mL). These two images show that concentrations above the MIC produce analogous remarkable effects on the bacterium: the bacterial envelope becomes strongly fragmented and completely loses its solidarity.

204

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

Fig 6. SEM images of bacteria at different concentration of SB006. A, Untreated bacterium. B, Treatment with 4 μg/mL of SB006 (MIC). C, Treatment with 8 μg/mL of SB006. D, Treatment with 64 μg/mL of SB006. SEM images of treated bacteria show the same morphological alterations already highlighted in AFM images, consisting in a progressive increase of envelope fragmentation.

in size and have a peculiar dendritic shape. In previous studies the inconvenience was eluded by washing the bacterial suspension in distilled water before depositing it on the substrate [26-28,36]. Considering this treatment to be unnecessary, we preferred to avoid it so as to limit any possible masking of SB006-induced alterations due to hypotonic conditions [36]. AFM images of the untreated sample clearly show the bacillary shape of the bacteria. The bacteria length ranges between 1.5 and 2.5 μm, and bacteria width is approximately 0.5 μm. Untreated bacteria have a relatively smooth and close-knit surface (Figure 5, A). Their average height is 207 ± 11 nm, which is consistent with what has been reported by Yao et al [43] for the same air-dried bacteria strain placed on aluminum oxide anodic filters. The comparison between AFM images of untreated and treated bacteria highlights the morphological alterations induced by SB006 treatment: treated bacteria are characterized by evident surface corrugation; the external surface is marked by ruptures and bulging. This dramatic change in morphology differs from the alteration of surface roughness conserving the original bacterial shape observed by da Silva et al [31] and Meinken

et al [35], whereas it resembles the extensive cell damage observed by Braga and Ricci [32] and Li et al [36]. These features first appear at a concentration of SB006 equal to the MIC (4 μg/mL) and become more evident at concentrations of SB006 equal to 8 μg/mL and 64 μg/mL. The initial damage consists in the collapse of the bacterium along its centerline (Figure 5, B) as reported by Sharma et al [33].

Table 1 Average bacterial heights (H) and average values of the bacterium envelope irregularity parameter (P) for each analyzed bacterial sample a SB006 concentration (μg/mL)

Bacterium average height H (nm)

Parameter P of the bacterium envelope irregularity

0

207 ± 11

1.75 ± 0.25

4

135 ± 1

2.50 ± 0.50

8

117 ± 7

4.17 ± 0.48

130 ± 5

5.25 ± 0.25

64 a

Irrespectively of SB006 concentration, for treated bacteria H becomes about half of the value that characterizes untreated bacteria. In contrast, the average parameter P shows a clearly increasing trend with the increase of SB006 concentration.

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

205

Fig 7. AFM images of a bacterium before and after an AFM-tip nanoindentation experiment. A, AFM image of the bacterium before force curves acquisition. The crosses on the bacterium surface indicate the positions of nanoindentation. B, AFM image of the bacterium after force curves acquisition.

AFM images (Figure 5, C and D) of sample treated with higher concentrations of SB006 (8 μg/mL, 64 μg/mL) reveal a complete collapse and loss of smoothness of the bacterium envelope. The same effects were evident in SEM images (Figure 6). This visible increase of membrane fragmentariness concurrent with the increase of SB006 concentration is quantitatively represented by the parameter P (see Materials and methods and Figure 2 for P calculation). As a matter of fact, the highest values reached by P are characteristic of the most morphologically altered bacteria (Table 1). These morphological alterations are coupled to a decrease of the average height H (see Materials and Methods section for H calculation). Indeed, as indicated in Table 1, the average height is 207 ± 11 nm for untreated cells and reduces of about

40 % for SB006 treated cells. Because in all the observations there is not a significant change in bacteria lateral size, the decrease in H corresponds to a loss of cell volume. This sounds reasonable and supports the morphological observations. The loss of cell volume may be due to a higher loss of cytoplasmic material during the dehydration step of the sample, being mediated by the increased permeability of the disrupted membranes of treated bacteria. Moreover, this hypothesis is supported by previous β-galactosidase release tests, which hinted to inner membrane permeabilization. Regarding the mechanical properties evaluation, because of the enhanced fragility of dehydrated biological materials [42,43], nanoindentation tests on dried bacteria led to unavoidable permanent deformations. Figure 7, which shows the same bacterium before and after a nanoindentation

Fig 8. AFM force-distance curves acquired on a bacterium and on bare mica. The bacterium behaves as a spring of kbact = 6.87 N/m. The hysteresis of the curve acquired on the bacterium (due to its permanent deformation) is evident. In contrast, hysteresis is absent on the curve acquired on mica (no permanent deformation). The adhesion force between the cantilever tip and the bacterium is equal to 48 nN. Measures were performed with a cantilever of spring constant equal to 8.13 N/m.

206

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207

experiment, is representative of the damage occurring on bacteria after such measurements. Nanoindentation measurements induced permanent deformation of the bacterium envelope. The effect is due to the fragility acquired by the biological material when dehydrated. Likewise, a typical force-distance curve registered on a bacterium and a typical force-curve registered on the rigid substrate are reported in Figure 8. The comparison highlights the reduced slope and the clear hysteresis that characterize force-distance curves acquired on bacteria. From the approach branch of the forcedistance curve, according to equation 2 we calculated the elastic constant of the bacterium, kbact, which is directly correlated with its mechanical properties. From the retraction branch of the force-distance curve we assessed the adhesion force acting between the bacterium and the cantilever tip. A typical force-distance curve collected on an untreated bacterium is shown in Figure 8. In the same figure a forcedistance curve collected on bare mica is also reported for reference purposes. In this reported case, kbact results were equal to 6.87 N/m, whereas the tip/bacterium adhesion force is equal to 48 nN (the cantilever spring constant is 8.13 N/m). To assess SB006 effects on bacterial mechanical properties, several nanoindentation experiments were performed for every peptide concentration. Then the mean elastic constant, Kbact, and the related uncertainty (taken as the standard deviation of the mean from different cells) were determined. For control bacteria Kbact was equal to 6.33 ± 0.62 N/m, whereas for treated ones Kbact was equal to 6.26 ± 0.91 N/m. The two values are consistent, indicating that the mechanical properties (e.g., rigidity) of SB006 are unaffected by the peptide treatment. This result supports the current membrane interaction hypothesis, according to which the peptide does not affect the bacterium wall, thus ensuring its rigidity. It is worthwhile noting that the estimation of the elastic constant is in good agreement with the literature data. The overall value of Kbact that results from our nanoindentation measurements in a dry environment is 6.30 ± 0.51 N/m. Such a value is one order of magnitude higher than the value that was determined for E. coli [33] and P. aeruginosa [41] by nanoindentation in a physiological (wet) environment. This ratio between dry and wet elastic constants is the same reported by Thwaites and Surana on B. subtilis [42], and Yao et al on E. coli [43]. Conclusions By means of AFM this research singled out morphological and mechanical alterations induced by different concentrations of the antimicrobial multimeric peptide SB006 on P. aeruginosa ATCC 27853. To obtain quantitative information from morphological images we developed a novel approach, based on the definition of a surface irregularity parameter, P, and of the related ON/OFF threshold. This allowed us to objectively state that morphological alterations due to SB006

consisted in fragmentariness and loss of solidarity of the membrane wall and in a severe decrease of the bacteria volume, the latter possibly being explained by leakage of cytoplasmic material. Bacterial mechanical properties were directly measured through AFM-tip nanoindentation. This showed that cell rigidity is not affected by the treatment with SB006, suggesting that SB006 does not act on the bacterial wall (which is responsible for the bacterium rigidity). Both of these sets of results helped us to elucidate the mode of action of SB006 and, consistently with previous β-galactosidase tests, support the membrane interaction hypothesis. AFM analysis procedures used in this investigation produced quantitative and consistent results, confirming AFM potentiality for gaining unique, direct insights into the action mechanisms of antimicrobial agents. Thus, it may be applied to evaluate bacterial alterations caused by antibiotics that work through different mechanisms of action. Indeed further investigation is needed. In particular the performance of the P parameter will be assessed by experiments on bacteria treated with other antibiotics and nanoindentation experiments checked on bacteria treated with antibiotics that are proved to affect cell wall structure and synthesis (i.e., rigidity). Acknowledgments The authors wish to thank Alessandro Rivetti for SEM imaging. This work was developed in the framework of LATEMAR (Laboratorio di Tecnologie Elettrobiochimiche Miniáturizzate per l'Analisi e la Ricerca). References [1] Hancock REW. Peptide antibiotics. Lancet 1997;349:418-22. [2] Wu M, Maier E, Benz R, Hancock RE. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 1999;38:7235-42. [3] Pereira HA. Novel therapies based on cationic antimicrobial peptides. CurrPharmacol Biotechnol 2006;7:229-34. [4] Zhang L, Falla TJ. Antimicrobial peptides: therapeutic potential. Exp Opin Pharmacother 2006;7:653-63. [5] Boman HG. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 1995;13:61-92. [6] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389-95. [7] Marcus JP, Green JL, Goulter KC, Manners JM. A family of antimicrobial peptides is produced by processing of a 7S globulin protein in Macadamia integrifolia kernels. J Plant 1999;19:699-710. [8] Jenssen H, Hamill P, Hancock REW. Peptide antimicrobial agents. Clin Microbiol Rev 2006;19:491-511. [9] Wang Z, Wang G. APD: the antimicrobial peptide database. Nucleic Acids Res 2004;32(Database issue):D590-2. [10] Dijkshoorn L, Brouwer CPJM, Bogaards SJP, Nemec A, van den Broek PJ, Nibbering PH. The synthetic N-terminal peptide of human lactoferrin, hLF(1-11), is highly effective against experimental infection caused by multidrug-resistant Acinetobacter baumanni. Antimicrob Agents Chemother 2004;48:4919-21.

G. Rossetto et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 198–207 [11] Mohammad FV, Noorwala M, Ahmad VU, Sener B. Bidesmosidic triterpenoidal saponins from the roots of Symphytum officinale. Planta Med 1995;61:94. [12] Aley SB, Zimmerman M, Hetsko M, Selsted ME, Gillin FD. Killing of Giardia lamblia by cryptdins and cationic neutrophil peptides. Infect Immun 1994;62:5397-403. [13] Hancock REW, Lehrer R. Cationic peptides: a new source of antibiotics. TibTech 1998;16:82-7. [14] Bracci L, Falciani C, Lelli B, Lozzi L, Runci Y, Pini A, et al. Synthetic peptides in the form of dendrimers become resistant to protease activity. J Biol Chem 2003;278:46590-5. [15] Pini A, Giuliani A, Falciani C, Runci Y, Ricci C, Lelli B, et al. Antimicrobial activity of novel dendrimeric peptides obtained by phage display selection and rational modification. Antimicrob Agents Chemother 2005;49:2665-72. [16] Tam PJ, Lu Y-A, Yang J-L. Antimicrobial dendrimeric peptides. Eur J Biochem 2002;269:923-32. [17] Giacometti A, Cirioni O, Barchiesi F, Fortuna M, Scalise G. In-vitro activity of cationic peptides alone and in combination with clinically used antimicrobial agents against Pseudomonas aeruginosa. J Antimicrob Chemother 1999;44:641-5. [18] Hancock REW, Chapple DS. Peptide antibiotics. Antimicrob Agents Chemother 1999;43:1317-23. [19] Zhang L, Dhillon P, Yan H, Farmer S, Hancock REW. Interaction of bacterial cationic peptide with outer and cytoplasmic membranes of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2000;44: 3317-21. [20] Epand RM, Vogel HJ. Diversity of antimicrobial peptides and their mechanism of action. Biochim Biophys Acta 1999;1462: 11-28. [21] Matsuzaki K. Why and how are peptide-lipid interactions utilized for self-defence? Magainins and tachyplesins as archetypes. Biochim Biophys Acta 1999;1462:1-10. [22] Alessandrini A, Facci P. AFM: a versatile tool in biophysics. Meas Sci Technol 2005;16:R65-R92. [23] Dufrêne YF, Müller DJ. Microbial surfaces investigated using atomic force microscopy. Methods Microb 2005;34:163-97. [24] Santos NC, Castano MARB. An overview of the biophysical applications of atomic force microscopy. Biophys Chem 2003;107: 133-49. [25] Bolshakova AV, Kiselyova OI, Filonov AS, Frolova OY, Lyubchenko YL, Yaminsky IV. Comparative studies of bacteria with an atomic force microscopy operating in different modes. Ultramicroscopy 2001; 86:121-8. [26] Robichon D, Girard J-C, Cenatiempo Y, Cavellier J-F. Atomic force microscopy imaging of dried or living bacteria. Life Sci 1999;322: 687-93. [27] Doktycz MJ, Sullivan CJ, Hoyt PR, Pelletier DA, Wu S, Allison DP. AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces. Ultramicroscopy 2003;97:209-16.

207

[28] Nishino T, Ikemoto E, Kogure K. Application of atomic force microscopy to observation of marine bacteria. J Oceanography 2004;60:219-25. [29] Umeda A, Saito M, Amako K. Surface characteristics of Gramnegative and Gram-positive bacteria in an atomic force microscope image. Microbiol Immunol 1998;42:159-64. [30] Kasas S, Fellay B, Cargnello R. Observation of the action of penicillin on Bacillus subtilis using atomic force microscopy: technique for the preparation of bacteria. Surf Interface Anal 1994;21:400-1. [31] da Silva A, Teschke O. Dynamics of the antimicrobial peptide PGLa action on Escherichia coli monitored by atomic force microscopy. World J Microbiol Biotechnol 2005;21:1103-10. [32] Braga PC, Ricci D. Atomic force microscopy: application to investigation of Escherichia coli morphology before and after exposure to cefodizime. Antimicrob Agents Chemother 1998;42:18-22. [33] Sharma S, Sen P, Mukhopadhyay SN, Guha SK. Microbicidal male contraceptive-risug induced morphostructural damage in E. coli. Colloids Surf B Biointerfaces 2003;32:43-50. [34] Peng L, Yi L, Zhexue L, Juncheng Z, Jiaxin D, Daiwen P, et al. Study on biological effect of La3+ on Escherichia coli by atomic force microscopy. J Inorg Biochem 2004;98:68-72. [35] Meincken M, Holroyd DL, Rautenbach M. Atomic force microscopy study of the effects of antimicrobial peptides on the cell envelope of Escherichia coli. Antimicrob Agents Chemother 2005;49:4085-92. [36] Li A, Lee PY, Ho B, Ding JL, Lim CT. Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria. Biochimica et Biophysica Acta 2007;1768:411-8. [37] Hassan A-E, Heinz WF, Antonik MD, D'Costa NP, Nageswaran S, Schoenenberger C, et al. Relative microelastic mapping of living cells by atomic force microscopy. J Biophys 1998;74:1564-78. [38] Bolshakova AV, Kiselyova OI, Yaminsky IV. Microbial surfaces investigated using atomic force microscopy. Biotechnol Prog 2004;20: 1615-22. [39] Boulbitch A. Deformation of the envelope of a spherical Gramnegative bacterium during the atomic force microscopy measurements. J Electron Microsc 2000;49:459-62. [40] Arnoldi M, Fritz M, Bäuerlein E, Radmacher M, Sackmann E, Boulbitch A. Bacterial turgor pressure can be measured by atomic force microscopy. Phys E Rev 2000;62:1034-44. [41] Yao X, Walter J, Burke S, Stewart S, Jericho M, Pink D, et al. Atomic force microscopy and theoretical consideration of surface properties and turgor pressure of bacteria. Colloids Surf B Biointerfaces 2001;23:213-30. [42] Thwaites JJ, Surana UC. Mechanical properties of Bacillus subtilis cell walls: effects of removing residual culture medium. J Bacteriol 1991; 173:197-203. [43] Yao X, Jericho M, Pink D, Beveridge T. Thickness and elasticity of Gram-negative Murein sacculi measured by atomic force microscopy. J Bacteriol 1999;181:6865-75.