Brief reports on the use of atomic force microscopy in visualization of Mycobacterium tuberculosis

Genady Zhavnerko; Parissa Farnia; Nikolai Poleschuk

doi:10.4103/bbrj.bbrj_21_17

REVIEW ARTICLE

Year : 2017 | Volume : 1 | Issue : 1 | Page : 9-13

Brief reports on the use of atomic force microscopy in visualization of Mycobacterium tuberculosis

Genady Zhavnerko¹, Parissa Farnia², Nikolai Poleschuk¹
¹ The Republican Research and Practical Centre for Epidemiology and Microbiology, Minsk, Belarus
² Mycobacteriology Research Centre (MRC), National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Web Publication

24-Jul-2017

Correspondence Address:
Genady Zhavnerko
The Republican Research and Practical Centre for Epidemiology and Microbiology, ul. Filimonova 23, 220016 Minsk
Belarus

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_21_17

Abstract

Background: By invention of the atomic force microscope (AFM) in 1986, the imaging of surfaces objects at nanometer-scale resolutions becomes possible. Although, in the beginning, AFM was applied almost exclusively to characterize the surfaces of nonbiological materials, at present the application of the AFM to biological and biomedical research has increased exponentially. Methods: In this study, we tried to investigate and visualize Mycobacterium tuberculosis under AFM. The transformation of bacterial shape and the formation of “hard” shell in resistant or dormant conditions were characterized and identified. Results: Application of AFM for the study of antibiotic-resistant forms of M. tuberculosis revealed the presence of round-shaped bacteria along with conventional rod-shaped ones. There has also been concluded changing of the surface charge of the cell membrane for mutated forms since round-shaped bacteria fixed on the charged surface less strongly and can be moved along the surface by a microscope tip with easily. Conclusions: In brief, this article highlights the optimal operation modes and base principal to study dangerous bacilli under AFM.

Keywords: Atomic force microscope, bacteria, mutated forms, Mycobacterium tuberculosis

How to cite this article:
Zhavnerko G, Farnia P, Poleschuk N. Brief reports on the use of atomic force microscopy in visualization of Mycobacterium tuberculosis. Biomed Biotechnol Res J 2017;1:9-13

How to cite this URL:
Zhavnerko G, Farnia P, Poleschuk N. Brief reports on the use of atomic force microscopy in visualization of Mycobacterium tuberculosis. Biomed Biotechnol Res J [serial online] 2017 [cited 2023 May 30];1:9-13. Available from: https://www.bmbtrj.org/text.asp?2017/1/1/9/211408

Introduction

Atomic force microscopy (AFM), invented in 1986,^[1] is one of the most effective tools for studying the structure and surface properties of biological samples, both for individual proteins and cell cultures. At present, AFM measurements become rather routine although the opportunities offered by optical microscopy methods to study biological objects have been limited not so long ago.

There are several scanning modes of AFM [Figure 1], so-called contact mode (CM) and tapping non-contact mode (TM), and regimes that allow to scan both in air and in liquid. It is believed that TM is more gentle, provides higher resolution, and has minimal influence on the sample surface during scanning. The advantage of the contact regimen is significantly higher scan rate, which is important when a large number of analyses take place. Each mode is intended for the decision of a certain number of the problems. It gives a lot of opportunities to the researcher and allows working in the mode, which is most pertinent and effective at given experimental conditions. Both scan modes were used in our research depending on the task although the CM in air had preference in view of obtaining contrast images in the friction regimen together with the information received on the adhesion of the probe of a tip to the surface during scanning of cell membrane.

Figure 1: Potential (f) of interaction of a probe with the sample depending on the distance (h) between tip and sample

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AFM is based on registration of deflections of the probe caused by various short- and long-range forces, existing between cantilever and a surface. The sum of the forces operating on various distances between the sample and cantilever [Figure 1] characterizes operating modes of AFM. The force of adhesion arising in a scope of molecular forces (Van der Waals attraction in the absence of contact and elastic interaction at contact) between pairs of molecules of a probe and the sample has the particular interest among the described cases of interaction cantilever and the sample when between one stream of molecules of a probe and the sample operate and between others, in this a case. The precise definition of interaction force of a probe with the sample is impossible in intermediate area between Van der Waals attraction forces (potential ~−1/r⁶) in the absence of contact and elastic interaction at a contact (pushing away at potential ~−1/r¹²).

It is necessary to distinguish two cases of adhesion: “a probe-liquid film on a surface” and the case of “a probe-solid sample.” If the first case is reduced to capillary interaction, the reason of adhesive forces between a probe and the solid sample is molecular electrostatic interaction.

Adhesion is no conservative process which can be defined quantitatively as the work that used for separation named work of adhesion (W):^[2]

Indexes mean d – dispersive interaction of London, P – a dipole–dipolar (orientation) interaction, i – induction interaction, h – hydrogen communication, π – π-communication, da – donor-acceptor communication, and e – electrostatic interaction. The combination of three first terms is the work of Van der Waals forces.

The approached models are used for the quantitative description of adhesion. Different approximations are used to solve of Hertz contact problem for solid bodies. Derjaguin-Muller-Toropov model^[3] is applicable for the probes with small radius of spherical tip with high rigidity. It is supposed that the geometry of the deformed surfaces closely approximated that gives the solution of Hertz problem. The account of Van der Waals forces of on perimeter of a contact platform leads to an additional attraction between a probe and the sample, which reduces forces of elastic retract.

Expressions for pressure (P) and penetration depths (h) are described by the following equations.

Where R is the tip radius of a probe, a is the radius of contact, W is the work of adhesion, and K is the effective modulus of elasticity.

In the equation 4, μ and μ₁ are the Poisson's ratio for AFM probe materials and the surface and E and E₁ are their modules of elasticity.

Johnson-Kendall-Roberts model^[4] is suitable for the probes with the major radius of curvature (even, most likely, for macroscopical bodies) and small rigidity. Such systems are called strongly adhesive. The model considers the contribution of Van der Waals forces within contact area. Attraction is arising at strong contact, which not only reduces force of elastic retract but also leads to negative force and “loop” formation [h < 0]. The “loop” describes the following formulae:

The main assumptions and restrictions within each theory are summarized in [Table 1], and the corresponding equations^[5] are presented in [Table 2].

Table 1: Comparison of quantitative adhesion theories

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Table 2: Normalized equations of quantitative adhesion theories

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Force (attraction/retraction) curves allow estimating adhesion, viscosity, surface contamination, a thickness of a layer, and also local variations in elastic properties of the surface.^{[3],[4],[5],[6],[7],[8],[9],[10]} “The attraction/retraction” curve represents the plot of measured console deviation dependence from scanner lengthening. The curves recorded in laboratory are specific to each sample; however, it is possible to distinguish the main peculiarities to characterize the sample by force spectroscopy mode of AFM.

In general, a place where new data are obtained by AFM is an interdisciplinary field including medicine, chemistry, physics, and biology. In particular, AFM can be used in medicine and biology to identify a variety of infectious agents or to confirm previously established diagnosis. AFM gives possibilities not only to visualize the morphology of a surface or the stages of cell evolution depending on the outer conditions but also to probe the separate places of a surface to estimate cell wall elasticity and rigidity. Microbial cell walls from different bacteria have already studied by AFM.^{[11],[12],[13],[14]} Peculiarities of microbial surfaces together with analysis of possible artifacts that arisen during scanning are described in the study by Burnham and Colton.^[11] Decreasing of cell wall stiffness due to treatment of the Staphylococcus aureus with antistaphylococcal agent (lysostaphin) was demonstrated in the study by Francius et al.^[13] The details of the structure of Pseudomonas aeruginosa Pili was also characterized by AFM.^[14]

Results and Discussion

Using AFM, we successfully could show an altered Mycobacterium tuberculosis modified under the influence of antibiotics or when they are stored under anaerobic conditions, as well as the characterization of structural features of the bacteria.^{[15],[16],[17],[18]} Furthermore, we showed that the success of cell cultures analysis by AFM depends on the methods of their fixation on the surface. Polyelectrolyte films, both anionic (poly styrene sulfonate [PSS]) and cationic (polyethylenimine [PEI]), are good for fixation of bacteria on the surface. To visualize tuberculosis bacilli by AFM, the cells have to be first immobilized by mechanical trapping^[19] onto isopore polycarbonate membrane (Millipore). After filtering a concentrated cell suspension, the filter will gently rinse with deionized water and carefully cut and attach to a steel sample puck (Veeco Metrology Group) using a small piece of adhesive tape. The other possibility is to use charged silicon surface. Silicon plates can be modified by alternative adsorption of 1 mg/ml PEI and PSS solutions during 15 min incubation. The treated surfaces become (negatively or positively) charged after last treatment with PSS or PEI, respectively. For AFM imaging record, the CM using Nanoscope 3-D Multimode AFM (Veeco, Santa Barbara, CA, USA) was used. This device is equipped with a < E > calibrated scanner using the manufacturer's grating. AFM images that obtained in CM and TM are 100 mm and 200 mm nanoprobe cantilevers (standard spring constants ranging from 0.06 to 0.52 N/m) with oxide-sharpened Si₃N₄ integral tips (Veeco NanoProbe Tips NP-20) were used for CM mode. Silicon cantilevers with resonance frequency ~260 kHz (Veeco NanoProbe Tips RTESP) are used for TM regimen. Generally, the TM of AFM increases resolution and do not disturb surface of the sample in polymer matrix [Figure 2]. As seen in [Figure 2], the encapsulated shell of M. tuberculosis is composed of several layers, whose thickness varies in different parts of bacteria, ranging from 12 to 60 nm. It is believed^[20],[21] that an inner layer, which is equal ~4 nm, contains peptidoglycan^[20] and more thick layer is hydrophobic and consists of mycolic acid, which covalently bound to arabinogalactan. There are polysaccharides, glycoproteins, and glycolipids in an outer layer of M. tuberculosis cell wall that confers resistance to bacteria and ensures their life activity. Earlier,^[16] the presence of oval-shaped bacteria was demonstrated by TEM in analyzed culture. Unfortunately, it is quite difficult to distinguish round shaped forms from cross-section of rod-like ones on the samples in polymer matrix due to noncontrolled orientation of M. tuberculosis. That is why AFM search of round-shaped forms was carried out on planar surfaces. There is no doubt that the outer shell of M. tuberculosis may be charged due to the presence of glycoproteins on the surface of the cell membrane. Thus, M. tuberculosis can be fixed on planar surface electrostatically. Hence, M. tuberculosis can be fixed on the planar surface electrostatically using silicon substrate with polyelectrolyte layer [Figure 3].

Figure 2: Truncated surface from Mycobacterium in paraffin, obtained by atomic force microscope in tapping mode

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Figure 3: Typical images of Mycobacterium on a silicon surface modified with positively charged polyethylenimine film

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Usually, the friction image in CM of AFM (so-called lateral force microscopy) gives more contrast and allows the detailed structure of surface to be seen. Different colors in different parts of the image visualize a different “hardness” of different places of a surface. Several interesting peculiarities can be distinguished, in particular, a feature that marked by arrow look-like the sharp “spine” under the cell wall membrane in [Figure 4]c (marked by arrow). Indeed, in friction image different places of M. tuberculosis showed different adhesion of a tip of AFM to a surface [Figure 4]. Adhesion of AFM tip to the surface of a cell under dividing is much lower (at least 2–3 times) than that for “old” shell of a bacterial cell. Taking into account sufficiently hydrophilic character surface of a tip and capillary forces, we can assume with high probability that “old” shell bacteria is more hydrophilic in comparison with the part under dividing. “Approach-retraction” hysteresis curve of AFM tip to the surface of the polycarbonate filter is less than that for the case of the tested bacterial cell. First, it proves hydrophilic character of bacterial wall in comparison with hydrophobic surface of membrane. Second, it explains sufficiently high mobility of the bacteria on the filter surface. Search of more suitable surface for stable fixation of cells onto surface was carried out to avoid distortion of bacteria during AFM analysis. The silicon surface modified with polyelectrolyte film was selected for this purpose. Recently, an approach of modification of a smooth mica surface with gelatin film was successfully used for immobilization of both Gram-positive (S. aureus) and Gram-negative (Escherichia coli) bacteria.^[22] Onto the contrary, we also showed the good immobilization of M. tuberculosis on surface of silicon modified with both PEI and PSS sublayers [Figure 5]. The image of bacteria was high-quality images without distortion. Therefore, the success of AFM analysis of cell cultures by AFM depends on methods of imaging, cell preparation, and methods of fixation on the surface.

Figure 4: The stages of division of bacteria: (a) initial, (b) immediately before cell division, (c) retraction nearly half of the almost divided bacilli in the pore of the filter with the curves “adhesion/retraction” for different places of the surface

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Figure 5: Atomic force microscope image of the Mycobacterium tuberculosis after ½ year storage in anaerobic conditions: (a) adsorption on the surface of polyethylenimine sublayer; (b) on poly (styrene sulfonate) sub.layer

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Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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