1.

Introduction

Amoeba is one of the protozoa that exists ubiquitously in nature and can be found commonly in soil and water sources.¹ Several amoeba species can cause diseases, including the ones leading to the death of humans and animals,^2,3 such as Naegleria fowleri, which causes amoebic meningoencephalitis; Acanthamoeba spp., and Balamuthia mandrillaris, resulting in amoebic encephalitis.^2,4 Better understanding of the amoeba life cycle and amoebic infection development can assist in epidemiological monitoring, as well as in the diagnosis and treatment of the diseases.

Amoeba in its life cycle can exist in two main forms, the cyst and the trophozoite (trophic amoeba). When the environment does not provide suitable survival conditions, amoeba loses its pseudopodia movement and generates a protective wall to form the cyst, which is immotile and metabolically inert. When the environment becomes favorable for the amoeba survival, the cyst activates, sheds its coat, and becomes trophic amoeba in the process of excystation.⁵ The cyst in the dormant state is more resistant to disinfection treatment than trophozoite, as has been shown for the cyst stage of Acanthamoeba spp. It has also been found resistant to extreme physical and chemical conditions.^2,5,6 To date, research on the amoeba life cycle has been mostly focused on the encystation (the cyst forming), and characteristic signaling pathways have been described.^7,8 Until recently, however, the biochemical and molecular mechanisms involved in the transitions from cyst to trophic form and pathways controlling this process were not fully studied and understood.

Raman spectroscopy provides information about the chemical structure and could be a useful tool for studying the life cycle of amoeba. Although laser technologies have been widely used in environmental and biomedical sciences, no information has been published on Raman spectroscopic studies of amoeba.^9,10 With its molecular specific nature, Raman shifts can serve as fingerprints for identification of the molecular composition and structure and provide information for the characterization of the state of biological samples. The non-invasive advantage renders the possibility for investigation in living samples. Raman spectroscopy has been successfully applied in cell division, cell death, respiration, and other metabolic processes via Raman spectral analysis of the compositional and structural changes of the cellular components.^11–14 New methods based on Raman spectroscopy (such as surface-enhanced Raman scattering, coherent anti-stokes Raman scattering, Raman mapping, etc.¹⁵) have been developed recently.^9,10 For example, Raman mapping can provide information about the distribution of chemical species in the sample;^16–18 time- and space-resolved image analysis yields dynamical and distribution information on the studied samples.^19,20

In this work, we present the Raman spectral analysis of amoeba (Acanthamoeba polyphaga) in the cyst stages. The changes of the relative intensities of characteristic cellular Raman bands, with a focus on carotenoids spectral components during the amoeba cyst activation and excystation progression are analyzed. The strong correlation between the carotenoid signals and the life cycle stages are observed. The analysis of Raman spectra allows for developing the Raman spectroscopic applications for detection of amoeba in the virulent state and providing a rapid test of amoebic inflammation of environmental mediums, as well as assisting in the early/express diagnostics and treatment of an infection.

2.

Materials and Methods

3.

Results and Discussion

The amoeba representatives are the Acanthamoeba polyphaga. As for its pathogenecity, Acanthamoeba can cause amoebic keratitis, the vision-threatening infection of the cornea that usually occurs in contact lens wearers.² In this study, we use Acanthamoeba as a model to discuss the Raman signature of the life cycle of this microorganism. The well-known amoeba life cycle stages^2,3,21 observed using microscopic optical imaging were compared with the Raman spectroscopic results. The Acanthamoeba polyphaga has two main states in its life cycle, a trophozoite stage and a double-walled cyst stage. The cell has a single vesicular nucleus and the nucleus has a centrally placed, large, densely staining nucleolus.² The Acanthamoeba in the trophozoites state ranges in size from 15 to $30\mu \mathrm{m}$ and the cyst size is $\sim 15\mu \mathrm{m}$.³ When food becomes scarce or subject to environmental stresses, the amoeba forms a protective spherical cell wall, decreases its metabolism, and becomes encysted. After completion of encystation, the amoeba is in a resting or stable mature cyst form. When the mature cyst encounters an optimal growth environment, the intercellular processes start to transform the cyst to the activated form, with increasing intercellular motility following detachment of the amoeba from the cyst wall and free amoeba motion inside the cyst envelope. Finally, the amoeba breaks out the cyst envelope as a trophozoite and leaves the empty envelope (cell wall). During the excystation processes, the transportation mediators play a very important role for transformation from a dormant/resting to an active metabolic state.²¹

In Fig. 1, the amoeba images during the encystation and excystation are presented. To quickly observe the amoeba encystation process, the temperature of the medium in the culture dish was decreased to 4°C. Figure 1(I) depicts amoeba images of the transformation from the trophozoite to the cyst stage as a result of the temperature decrease. One can observe amoeba loses its characteristic pseudopoidal movement and generate a protective wall. Once the appropriate environment is established, the cysts shed their coat and resume trophic or vegetative life through an excystation process, as illustrated in Fig. 1(II). The cysts shown in Fig. 1(II) were obtained as a consequence of the depletion of medium nutrients. To initiate and to observe the excystation, the medium in the culture dish with the cysts was refreshed. After the cysts were exposed to optimal growth conditions, the transformation to the mature cyst stage began. The corresponding images during this process are presented in Fig. 2(I). It is known that this process is characterized by the increasing cytoplasmic motility between the center and periphery of the cyst, involving lipid granules, contractile vacuole, and other subcellular structures.²¹ The enhanced cytoplasmic movement facilitates the chemicals’ transfer in cytoplasm and the exchange between the endocyst and the plasma membrane, particularly some proteins and the RNA increase observed during the excystation.^5,21

Fig. 1

Observation of amoeba life cycle using optical (differential interference contrast) images: (I) amoeba encystation and (II) amoeba excystation.

Fig. 2

Mature cyst transformation to activated cyst stage: (I) optical images and (II) Raman spectra of mature cyst transforming to activated cyst stage. The spectra intensities are normalized to $1007{\mathrm{cm}}^{-1}$ band. (III) Dependences of Raman peaks intensity on time at cyst activation.

We try to observe the relocation of these substances and chemical changes using Raman spectroscopy. The laser beam was focused in the center of the cyst and the position was fixed during these measurements. The Raman spectra, corresponding to each stage in Fig. 2(I), are displayed in Fig. 2(II). The spectral bands were assigned according to the literature;^{19,20,22–24} characteristic Raman bands for living cells were observed. The band near $1660{\mathrm{cm}}^{-1}$ is the Amide I band from the $\mathrm{C}═\mathrm{O}$ of the $\alpha$-helix secondary structure of protein, with possible input of the $\mathrm{C}═\mathrm{C}$ stretch vibrations of phospholipid. The band near $1450{\mathrm{cm}}^{-1}$ corresponding to $\mathrm{C}─\mathrm{H}$ bending modes can also come from both protein and lipid. The band at $2930{\mathrm{cm}}^{-1}$ is assigned to $\mathrm{C}─{\mathrm{H}}_2$ symmetric and asymmetric stretch vibrations of the lipid components. The band at $1264{\mathrm{cm}}^{-1}$ as well as the peak at $3025{\mathrm{cm}}^{-1}$ are for $═\mathrm{C}─{\mathrm{H}}_{\mathrm{cis}}$ stretches from lipids and alkyls.^22,25 The lipids’ bands provide signals from the membranes and vesicles organelles which form part of the endosomal system. The changes of relative intensities of these bands are observed and can reflect metabolic activation, as the endosomal system is a mediator in membrane transport processes.^20,23

Intense Raman bands at $1007{\mathrm{cm}}^{-1}$, $1156{\mathrm{cm}}^{-1}$, $1519{\mathrm{cm}}^{-1}$, $2170{\mathrm{cm}}^{-1}$, $2315{\mathrm{cm}}^{-1}$, and $2670{\mathrm{cm}}^{-1}$ are observed in the cyst, but decrease at cyst activation. The band at $1007{\mathrm{cm}}^{-1}$ is usually attributed to phenylalanine ring breath for most living cells,¹⁵ but in amoeba, it is also accompanied by 1156 and $1519{\mathrm{cm}}^{-1}$ bands. These three bands together indicate the presence of carotenoids, characteristic of skeletal ${\nu }_3$, ${\nu }_2$, ${\nu }_1$ vibrations.^26–28 Weaker peaks an 2017, 2315, and $2670{\mathrm{cm}}^{-1}$ can also be attributed to carotenoids.²⁴ Carotenoids form a large group of isoprenoid pigments. They are synthesized by all photosynthetic organisms and many nonphotosynthetic bacteria and fungi.^14,29 Carotenoids exhibit an intense and well-developed Raman spectrum. The excitation by a 488-nm wavelength laser provides resonant enhancement of the carotenoid Raman signals;³⁰ thus, we observed the predominance of carotenoid peaks over proteins’ and lipids’ bands. It has been shown that the Raman signal of carotenoid can be used as a marker, and a method for identifying microorganisms by Raman spectroscopy has been proposed.³¹ There have been neither studies about carotenoids in amoeba nor amoeba’s Raman studies previously reported. However, the above listed bands can be well-assigned to carotenoid components. There has been increasing evidence showing the beneficial effects of carotenoid antioxidants in the human body.³⁰ Carotenoids in biological systems have important functions based on their ability to absorb light and prevent oxidation both in carotenogenic microorganisms and plants and in organisms that are not carotenogenic themselves but obtain carotenoids from food or medicines. Hundreds of carotenoids have been identified, studied, and used. In this study, we find this signal has a strong correlation with the life cycle of the amoeba. Thus, the observed variation on carotenoids in amoeba provides some information of the life cycle of amoeba with the carotenoid.^30–32

Figure 2(III) shows changes of the characteristic band intensities in the process of the cyst activation. From Figs. 2(II) and 2(III), one can see that Amide I Raman band is well detected at the activated cyst stage. It suggests that protein and RNA content increase during excystation.⁵ Simultaneously, a broad band centered at $2930{\mathrm{cm}}^{-1}$ assigned to aliphatic groups (CH vibrations in ${\mathrm{CH}}_2$, ${\mathrm{CH}}_3$ groups, mostly belonging to lipids) increases significantly, while the intensities of the bands of carotenoid components decrease. If we take a ratio of the 1519 to 2930 and then the 1156 to 2930 bands, we can show that the carotenoid decreases while the lipids increase with activation of the cyst (data not shown).

Raman spectra from the cystine activated stage (a) and from the cyst envelope after excystation (b) are compared in Fig. 3. In contrast to the image of a mature cyst [Fig. 4(a)], the trophozoite body inside the cyst envelope can be observed after cyst activation [Fig. 4(b)]. In the spectrum measured from a cyst in an activated state, both carotenoid’s Raman bands and characteristic bands for most living cells are presented, such as the Amide I and lipids. The optical image of the cyst envelope after excystation is shown in Fig. 4(d). For the cyst envelope, the intensity of the Raman signal measured at the same conditions as the spectrum before excystation significantly decreases; only bands corresponding to carotenoid components remain observable.

Fig. 3

Raman spectra of activated cysts and cysts’ envelopes. The spectra, not normalized, are measured under the same conditions focusing on the center of the cyst or envelope. After the amoeba’s excystation, the cyst’s envelope’s spectrum only reveals $\mathrm{C}─\mathrm{H}$ ($1156{\mathrm{cm}}^{-1}$) and $\mathrm{C}═\mathrm{C}$ ($1519{\mathrm{cm}}^{-1}$) signals, corresponding to the components from carotenoid.

Fig. 4

Optical images of (a) mature cyst; (b) activated cyst with trophozoite inside; (c) amoeba trophozoite; and (d) empty cell wall (cyst envelope) after excystation. (b) and (d) correspond to spectra (a) and (b) of Fig. 3.

The laser power in the measurements (in the focal spot) was $\sim 0.1\mathrm{mW}$ to avoid heating and photo-damage of the amoeba. The safety limit of light exposure becomes important in the study of living cells. The safety limits differ for various objects and strongly depend on experimental conditions, i.e., the lasers.³³ In addition to safety, the tasks of laser destruction or inactivation of biological objects, such as pathogenic microorganisms,³⁴ cancer cells,³⁵ etc., have been discussed. In this work, we demonstrate the laser treatment’s ability to inactivate the amoeba and to prevent its transformation to the active form. The cyst was irradiated with a CW laser of 488-nm wavelength and a relatively high power of 4 mW on focal spot for 45 min. The Raman spectra measured sequentially during the 45 min of continuous irradiation of the single cyst are shown in Fig. 5(a). We observed a decrease of the absolute intensity of the spectral bands with this treatment. In the first measurement, only carotenoids and probably phenylalanine (which we cannot separate from carotenoid’s peak) peaks are observed. After 5 to 10 min of exposure, a decrease in the bands of the carotenoids with an increasing exposure time was observed. The dependence of intensity on exposure time is shown in Fig. 5(b). For cyst after irradiation, no activation and corresponding chemical changes can be observed, particularly, the proteins signal is not increasing. Therefore, no transformation and excystation happened. Thus, the cyst is significantly damaged or killed by the laser irradiation without significant visible changes in the cyst morphology.

Fig. 5

Laser power effect on the cysts: (a) spectra measured with high laser power (4 mW) exposure for 45 min from the same cysts; spectra are not normalized; the focus point position and the exposure and measurement conditions did not change. (b) Dependence of the carotenoid’s Raman peaks intensity on laser exposure time.

To obtain and analyze the information about the space distribution of chemical components, Raman mapping is performed. In Fig. 6, an optical [Fig. 6(I)] image of cyst is compared to Raman mapping [Figs. 6(II) and 6(III)]. For these measurements, the medium with cysts was dropped and dried on an Si substrate. In Fig. 6(II), the spatial distribution of the Raman signal intensity from the cyst at $1519{\mathrm{cm}}^{-1}$ [Fig. 6(II-a)], carotenoid, and $3025{\mathrm{cm}}^{-1}$ [Fig. 6(II-b)], mostly from $═\mathrm{C}─\mathrm{H}$ in lipids, are presented. Corresponding three-dimensional Raman mapping is presented in Fig. 6(III). The predominant localization of the carotenoids in the amoeba cyst in the peripheral area adjacent to the cell wall, as well as the distribution of some lipid species, is observed. The enhanced cytoplasmic motion in the activated cyst between the cell’s center and the layer of lipid granules near the plasma membrane has been reported.²¹ The observed carotenoids in the cell periphery suggest their colocalization with the lipid granules, i.e., their involvement in cyst activation processes.

Fig. 6

Raman mapping of mature cysts: (I) optical image; (II) the mapping of the intensity of bands (a) at $1519{\mathrm{cm}}^{-1}$ (the signal is locked at 1510 to $1530{\mathrm{cm}}^{-1}$) and (b) at $3025{\mathrm{cm}}^{-1}$ (the signal is locked at 3000 to $3100{\mathrm{cm}}^{-1}$); (III) three-dimensional mapping of the same peak intensities: (a) $1519{\mathrm{cm}}^{-1}$ and (b) $3025{\mathrm{cm}}^{-1}$.

Raman spectroscopy has recently been applied for detection of microorganisms in biological and environmental samples. For this, carotenoid signals can serve both for the detection of the microorganisms (bacteria, fungi, microalgae, etc.) and for their discrimination.^14,25,31 Our results demonstrate the ability of Raman spectroscopy, and particularly, Raman mapping, for practical applications; it is possible to detect and identify amoeba's presence using carotenoid bands for imaging. The discrimination of its different stages in its life cycle can be important for the estimation of epidemiological situation and for diagnostics and prognosis of amoebic inflammation.

4.

Conclusion

In this report, a Raman spectroscopic study on a single amoeba has been performed. Raman spectra of the amoeba were measured and the spectral transformation corresponding to chemical changes during the amoeba cyst activation and excystation were observed. The Raman characteristic bands evidenced the carotenoid presence in the amoeba. The location of the carotenoid suggests their important roles in the amoeba excystation. Our results demonstrate the possibility for developing Raman applications for amoeba detection in environmental and biological mediums based on carotenoid signals, for identification of the stages of the amoeba life cycle and for the monitoring of the corresponding chemical and structural changes. Additionally, the effect of laser irradiation on the amoeba (in a cyst stage) viability is analyzed in an effort to develop methods for amoeba inactivation.