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In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets
Experimental Parasitology 210 (2020) 107842
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In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets
T
Johan Alvarado-Ocampoa,b, Lissette Retana-Moreiraa,b, Elizabeth Abrahams-Sandía,b,∗ a b
University of Costa Rica, Faculty of Microbiology, Department of Parasitology, San Pedro, San José, Costa Rica Centro de Investigación en Enfermedades Tropicales, San Pedro, San José, Costa Rica
A R T I C LE I N FO
A B S T R A C T
Keywords: Acanthamoeba Hemolytic activity Platelet aggregation Pathogenicity factor
Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperaturedependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.
1. Introduction Free-living amoebae (FLA) of the genus Acanthamoeba are widely distributed microorganisms that can be isolated from natural or artificial water bodies, drainages, sediments, air conditioning filters, surgical instruments, contact lenses, dialysis units, nasal cavities and skin lesions (Khan, 2006). They are considered emerging parasites, due to the increase and difficult handling of cases. In addition, they are known as amphizoic microorganisms, due to their ability to inhabit both external environments and the interior of living organisms, acting as a facultative parasite (Oddó, 2006). Acanthamoeba has been implicated in the development of severe infectious conditions such as amoebic granulomatous encephalitis and amoebic keratitis (infection of the cornea). Through molecular techniques, it has been determined that most of the isolates from human and environmental infections belong to the T4 genotype (Maciver et al., 2013). Studies regarding the main pathogenicity factors associated with these microorganisms allow the understanding of the mechanisms of
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damage the amoebae employ during the infection process. In Acanthamoeba, these factors can be of indirect origin (ubiquity, biofilm formation, resistance to therapeutic drugs, and physiological tolerance to osmolarity and temperature), which do not correspond to mechanisms that are activated to inflict damage, but that facilitate the survival of the amoeba in the pathogenic process (Lorenzo-Morales et al., 2015). On the other hand, there are also direct factors involved, which are both dependent and independent of contact. Contact-dependent factors are based on the adhesion of the amoeba to the tissues of the host as the first and most important step in the pathogenesis process. Here, extracellular matrix binding proteins are involved. In addition, processes such as phagocytosis and the release of harmful products such as the MIP-133 protease are activated (LorenzoMorales et al., 2015; Panjwani, 2010). Among the independent contact factors are soluble secretion products such as serine proteases, cysteine proteases, and other hydrolytic enzymes. These generate cytopathic effects that lead to cell death, the degradation of the extracellular matrix and the plasma membrane, with the eventual invasion of the
Corresponding author. University of Costa Rica, Faculty of Microbiology, Department of Parasitology, San Pedro, San José, Costa Rica. E-mail address: [email protected] (E. Abrahams-Sandí).
https://doi.org/10.1016/j.exppara.2020.107842 Received 3 December 2019; Received in revised form 10 January 2020; Accepted 18 January 2020 Available online 21 January 2020 0014-4894/ © 2020 Elsevier Inc. All rights reserved.
Experimental Parasitology 210 (2020) 107842
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Germany) and collected. The ACM was employed immediately after each collection process.
parasite to adjacent areas, promoting the persistence and aggravation of the infection (Panjwani, 2010; Khan et al., 2000) The damage mechanisms described above may involve cells such as erythrocytes and platelets (Mattana et al., 2009). Thus, hemolysis and platelet aggregation are factors that are also associated with pathogenicity and may represent a source of food for FLA or a direct target to carry out a successful invasion and the permanence inside the host. In other protozoa such as Entamoeba histolytica, this hemolytic capacity has been demonstrated (Lorenzo-Morales et al., 2015). Regarding Acanthamoeba, there are few studies related to the effects of Acanthamoeba on hemodynamics (Mattana et al., 2009; Nacapunchai et al., 2005). The description of clinical cases suggests that thrombosis in the blood vessels of the retina, necrotizing vasculitis (Awwad et al., 2007; Palme et al.,2017), hemorrhages and thrombi in the vessels of the periventricular tissue and the cerebellum, as well as other microcirculatory disorders (Feingold et al., 1998), could develop during a keratitis and a cerebral amebiasis. However, the mechanisms that generate this phenomenon are not yet clear and require further studies. Due to the scarcity of information in this area, the aim of this study was to evaluate the capacity of damage to some environmental isolates of Acanthamoeba and its products over blood elements.
2.3. Acanthamoeba trophozoite crude extracts Trophozoites of Acanthamoeba were suspended in phosphate buffer (PBS) (Sigma-Aldrich, St. Louis, USA) and 3 mL of each sample was centrifuged for 5 min at 5000 rpm. The pellet was resuspended in 1 mL of PBS and sonicated (10 cycles of 2 min each cycle) in an Ultrasonic Homogenizer (Cole-Parmer® Instrument Co., Illinois, USA). Finally, the samples were centrifuged at 7000 rpm for 6 min and filtered through a 0.22 μM pore membrane and the supernatant was collected. The crude extracts were employed immediately after each filtration process. 2.4. Hemolytic activity of live trophozoite, ACM and crude extracts over a suspension of erythrocytes 2.4.1. Live trophozoites For this assay, the protocol described by Mattana et al. (2009) was employed, with some modifications. Briefly, two suspensions were prepared: 1) a suspension of human erythrocytes in PBS (6 × 106 cells/ mL), and 2) a suspension of amoebae in PBS (3 × 106 amoebae/mL). The same volume of each suspension was added to 1.5 mL tubes (Eppendorf®, Hamburg, Germany), mixed and incubated for 0, 60 and 120 min at 37 °C. After this time, the samples were centrifuged for 10 min at 3000 rpm and the absorbance was measured in an UV-1800 spectrophotometer (Shimadzu® Corporation, Tokyo, Japan) at 546 nM. The blank solution employed was a suspension of erythrocytes in PBS (3 × 106 cells/mL) and the positive control (100% hemolysis) was a suspension of erythrocytes in distilled water (3 × 106 cells/mL). The hemolysis percentage was calculated as follows:
2. Material and methods 2.1. Amoebae The environmental isolates were characterized by the “Laboratorio de Protozoología Médica” of the University of Costa Rica (Table 1) (Castro-Artavia, 2015; Vargas-Ramírez, 2016) They were maintained in 25 cm2 cell culture flasks (Becton Dickinson®, New Jersey, USA) with peptone 2% (m/V), yeast extract 0,1% (m/V) and glucose 1,8 (m/V) massive growth medium (PYG), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10 μg/mL gentamicin (Sigma-Aldrich®, St. Louis, USA). Also, the evaluation of the environmental strain of Acanthamoeba castellanii Neff was included.
Hemolysis (%) = [Abstube – Absblank / Abscontrol+] * 100 Each assay was performed in triplicate. 2.4.2. ACM and crude extracts The activity of ACM and the crude extracts of trophozoites over erythrocytes were evaluated following the methodology described by Hraoui-Bloquet et al. (2014) with some modifications. Briefly, 100, 150 and 175 μL of a 30% suspension (V/V) of human erythrocytes in sterile saline solution 0.85% (m/V) were placed in Nunc™ 96-well ELISA plates (Thermo Fisher Scientific®, Massachusetts, USA). Then, 100, 50 and 25 μL of crude extract and ACM obtained after 24 h and 96 h of incubation were added, to obtain a final volume of 200 μL. The incubations were performed at 4, 30 and 37 °C and the absorbance of each well was measured in a Synergy HT plate reader (BioTek® Instruments Inc., Vermont, USA) at 540 nM. A lysate of erythrocytes in distilled water was employed as the positive control (100% hemolysis) and the
2.2. Acanthamoeba conditioned medium (ACM) To obtain the Acanthamoeba conditioned medium, the methodology described by Iqbal et al. was employed (Iqbal et al., 2014), with some modifications. Briefly, the PYG medium was removed from the culture flasks (with 1 × 106 amoebae/mL) and replaced with 3 mL of RPMI1640 (Sigma-Aldrich®, St. Louis, USA) with glucose 2% (m/V) (Merck Group, Darmstadt, Germany). Then, different amoebae cultures were incubated for 24 and 96 h at 30 °C and 2 mL of the conditioned medium was collected at the end of these times. Once collected, the medium was promptly centrifuged for 7 min at 3000 rpm, filtered through a 0.22 μM Minisart® syringe membrane (Sartorius Stedim Biotech, Goettingen,
Table 1 Axenic environmental isolates employed in these assays and their main characteristics related to pathogenic potential. Isolate
CSU6 CSU7 CSU8 CSU14 CSU18 DU3 DU13 H6 CSU7a H7
Sampling site
CSU: eyewash head CSU: eyewash sprayer CSU: eyewash sprayer CSU: eyewash head CSU eyewash sprayer Dental unit 3, Triple function syringe Dental unit 17, Triple function syringe Emergency Room: work table CSU powder Hospital water sample
Genotype
T4 T4 T4 T4 T4 T4 T4 T4 T4 T5
Cytopatic effect
Mild None Moderate Moderate High Mild Mild High Mild High
Thermotolerance*
Osmotolerance**
37 °C
40 °C
0, 5 M mannitol
1,0 M mannitol
+ + + + + + + + + +
+ – + – – + + + + –
+ + + + + + + + + +
+ – + + + + + + + –
Thermotolerance: proliferation at a 37 °C and 40 °C. **Osmotolerance: proliferation at different manitol concentrations, 0.5 M and 1.0 M. CSU: Combination shower unit. 2
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negative control included a suspension of erythrocytes in RPMI-1640 glucose 2% (m/V) or PBS. The percentage of hemolysis was calculated using the following formula:
Table 2 Percentage of hemolysis of a suspension of erythrocytes obtained for ten environmental isolates of Acanthamoeba and the Neff strain, at two different time points.
Hemolysis (%) = [Abswell – Abscontrol- / Abscontrol+] * 100
Isolate
Each assay was performed in triplicate. 2.5. Erythrophagocytosis assay
Neff CSU6 CSU7 CSU8 CSU14 CSU18 DU3 DU13 H6 CSU7a H7
2.5.1. Co-incubation of amoebae and erythrocytes Suspensions of erythrocytes (2 × 103 cells/μL) and amoebae (1 × 103 amoebae/μL) were prepared and the same volume of each suspension (cell:amoebae in a ratio 2:1) were placed in 96-well plates, incubated for 10 min at room temperature and then incubated for 60 min at 37 °C in a multimodal reader microscope Cytation (BioTek® Instruments Inc., Vermont, USA). Photographs of the samples were captured every 30 s and then analyzed in search of adhesion, phagocytic or lytic activity.
Average hemolysis percentage (%) ± SD 60 min
120 min
32.0 ± 0.7 24.4 ± 2.0 7.7 ± 2.2 6.4 ± 0.7 5.1 ± 1.3 2.6 ± 0.7 16.7 ± 3.9 5.1 ± 1.5 6.4 ± 2.7 1.3 ± 0.0 3.8 ± 0.7
42.3 41.0 24.4 15.4 39.7 16.7 26.9 16.7 35.9 18.0 25.6
± ± ± ± ± ± ± ± ± ± ±
2.0 1.3 1.5 2.7 2.2 3.2 2.0 0.7 3.4 0.7 0.7
CSU7, CSU8, CSU14, CSU18, DU13, H7 and H6 presented hemolysis percentages lower than 8% during the first hour and rose over 15% at the 2-hrs incubation period, while Neff, DU3 and CSU6 registered percentages greater than 15% in both incubation periods. Moreover, 80% of the isolates were able to produce a visible hemolytic effect over blood agar plates at the 5th-6th day of incubation. In this case, two patterns of hemolysis were registered: double halo (CSU6, H6 y CSU18) and another pattern similar to bacterial beta hemolysis (Neff, H7, CSU7, CSU14, DU3 y DU13) (Fig. 1). CSU7a and CSU8 showed results similar to the negative control. For this assay, it is important to highlight that, at 24–48 h of incubation, the encystment of most of the trophozoites placed over the agar plates was observed:
2.6. Hemolytic activity of live trophozoites, ACM and trophozoite crude extracts over blood agar plates In this case, the methodology described by Hraoui-Bloquet et al. (2014) was employed, with some modifications. Briefly, for the ACM and the crude extracts of trophozoites, 40 μL were placed in 7 mMdiameter wells inside 5% blood agar plates. In the case of complete trophozoites, a suspension of amoebae (1 × 106 amoebae/mL) in 50 μL PBS was placed on the surface of 5% blood agar plates. These plates were incubated at room temperature and 37 °C, with daily observation for one week. Controls of the assay included either PBS or RPMI-1640 with glucose 2% (m/V). The hemolytic activity of each sample was evaluated in quadruplicate. 2.7. Platelet aggregation assay ACM of each isolate was obtained as described above, with the following modifications: 1) PBS supplemented with 20 mM HEPES medium (Gibco®, Thermo Fisher Scientific®, Massachusetts, USA) was employed as the medium, and 2) amoebae were incubated for 2, 4 and 8 h at 30 °C prior to harvesting. Then, the samples were filtered through a 30 kDa membrane filter (Merck Millipore®, Darmstadt, Germany) and the supernatants were collected. The turbidimetric method described by Born and Cross (1963) was employed, with modifications. A volume of 5 μL of ACM (per each incubation time) was added inside the assay tube that contained an aliquot of 500 μL of platelet-rich plasma (PRP) (250,000 platelets/μL), to evaluate the increase in the passage of light and then create a platelet aggregation curve for a minimum of 5 min. An aliquot of 5 μL of 10 μM adenosine triphosphate (ADP) was employed as the positive control and an equal amount of PBS with 20 mM Gibco® HEPES medium was employed as the negative control. Also, a 500-μL aliquot of PRP (250,000 platelets/μL) was employed as the 0% transmittance solution, defined in the 490 4 + 4 optical aggregometer (Chronolog® Corporation, Pennsylvania, USA). The 100% transmittance solution corresponded to an equal aliquot of platelet-poor plasma (PPP). This assay was performed in triplicate. 3. Results No evidence of hemolytic activity was observed in the case of ACM and crude extracts of trophozoites, regardless of the incubation temperature and the type of assay performed. However, when the assays were performed employing live trophozoites, there was a marked hemolytic activity only at 37 °C. Hemolytic activity over erythrocyte suspensions was demonstrated in all of the isolates (Table 2). CSU7a,
Fig. 1. Effect of the living Acanthamoeba trophozoites over blood agar plates, after 6–8 days incubation at 37 °C: a. CSU6 (double halo pattern), b. CSU7 (simple halo pattern), c. CSU7a (no hemolisis). 3
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Fig. 2. Interaction between Acanthamoeba and a suspension of erythrocytes, during an incubation at 37 °C using light microscopy. Images were recorded at 15 min (a-b) and 45 min (c-d). Thick arrows indicate the multiple adhesion (a-c) and thin arrows show the deformation of the erythrocyte (c-d). Circles indicate the partial engulfment (c-d). Bar = 35 μM.
the limiting factor in their studies, both research groups refer to the low number of isolates assayed (only one or two). The aims of this work were to explore the hemolytic activity of ten environmental isolates of Acanthamoeba over human red blood cells, to assess the capacity of platelet aggregation by trophozoites through the production of the ADP agonist and to evaluate the phenomenon of erythrophagocytosis. All of the isolates employed were characterized by molecularly and physiologically in previous studies (Castro-Artavia, 2015; Vargas. Ramírez, 2016; Castro-Artavia et al., 2017) The results using erythrocytes in suspension showed a contact-dependent mechanism of hemolysis in all of the isolates. This was observed only when live trophozoites were used and at an incubation temperature of 37 °C, recording maximum activity at 2 h p.i. None of the conditioned media showed hemolytic activity. The above differs from the information published by Mattana et al. (2009), which reported, in addition to the activity of the live trophozoites, contact-independent hemolysis in the case of a clinical isolate of A. castellani. The authors proved the activity of soluble products secreted by the amoeba over erythrocytes in suspension, an activity that was Ca2+- and glucosedependent. More studies are needed to show if this haemolytic mechanism is exclusive of the clinical isolates. In the blood agar assay, only 80% of the isolates showed hemolytic activity. This may be related to the difference in the availability of the red blood cell in the agar plates, which could limit the phagocytosis and digestion of the cell by the amoebae. Another important aspect to be highlighted is that the hemolysis in the agar plates was observed until the 5th-6th day of incubation and at a temperature of 37 °C. The timelapse coincides with the encystment-excystment of trophozoites, which suggests that the effect observed in the plates could be the result of the release of products with hemolytic activity during these processes and it is not the direct destruction of the erythrocyte by the amoebae. Two different patterns of hemolysis were observed over the agar plates: a simple- (62,5%) or a double-halo (37,5%). According to the literature, the double halo hemolytic pattern has not been reported previously for these free-living amoebae, but a similar effect has been described in Streptococcus spp. In bacteria, this phenomenon is given by a non-synchronous production of hemolysins (Noble and Vosti, 1971) The possibility that the pattern observed in Acanthamoeba is linked to asynchronous mechanisms of secretion and diffusion of metabolites has
however, at 120–144 h p.i. They started the excystment process. In the erythrophagocytosis assay, the capacity of the amoebae to adhere to the erythrocytes was demonstrated, with the induction of cytoplasmic projections and morphological changes over these cells (Fig. 2). Erythrophagocytosis was also observed (Fig. 3). Finally, none of the tested ACM showed platelet aggregation capacity after 5 min of interaction with the platelet-rich plasma. According to the platelet aggregation curve, 0% aggregation was obtained for all of the samples tested. 4. Discussion The activity of Acanthamoeba over human erythrocytes has been poorly studied. In 2005, Nacapunchai et al. reported that environmental isolates with high pathogenic potential showed more adhesion to the erythrocyte than those with low pathogenic potential while Mattana et al. (2009) reported that these cells and platelets could be potential targets during the course of an Acanthamoeba castellani infection. In both studies, it was suggested that cell lysis occurs due to contact-dependent and -independent mechanisms that require the presence of some factors of the plasma and/or calcium ions and glucose. As
Fig. 3. Light microscopy image of an Acanthamoeba trophozoite with phagocytized erythrocytes after a 30 min incubation at 37 °C. Magnification: 40X, Bar = 40 μM. 4
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it is suggested that the platelet aggregation signal was not detected by the technique employed in this work, maybe due to ADP production at insufficient concentrations (< 5 mM).
not been ruled out and could be a subject for future research. In the present work, the erythrophagocytosis by Acanthamoeba was demonstrated, which contrasts with the findings reported by Nacapunchai et al. (2005) who did not observe the ingestion of red blood cells by the environmental isolates. As shown in Fig. 2, there was a process of adhesion of the amoebae to the erythrocytes, as well as a deformation of the red blood cell. However, the phagocytic index of all of the Acanthamoeba isolates was low, like the results reported by Shibayama et al. (2005). According to these authors, erythrophagocytosis is not a relevant mechanism in the pathogenesis of Acanthamoeba. Until now, most of the experiments that employ Acanthamoeba indicate that the contact-dependent mechanisms are the ones involved in the pathogenesis of the infection (González et al., 2013; FloresMaldonado et al., 2017), and the hemolytic activity is not the exception. It is not clear which factors or what is the exact mechanism the amoeba employs to lyse the erythrocytes. However, in the case of Acanthamoeba, there are reports of the secretion of proteases, lipases and the presence of some structures that, in other protozoan parasites, are related to the lysis of the red blood cells. For example, in Entamoeba histolytica, the hemolytic capacity is related to the presence of Ca2+dependent cytoplasmic phospholipases (González-Garza et al., 2000), while Naegleria fowleri secrete dense granules (in a contact-dependent way) that are associated to proteolysis and damage to the extracellular matrix (Chávez-Munguía et al., 2014). Phospholipases, serine- and cysteine-proteases and dense granules located near the ostiole have been described in Acanthamoeba (Chávez-Munguía et al., 2005; Silva-Caumo et al., 2014), so their role in the hemolytic activity of the amoeba could be subject to research. Our results showed that there is not an association between the level of cytopathic effect in vitro and the hemolytic activity since the isolates with a cytopathic effect reported as “none” or “mild” over the cell monolayer reached percentages of 15–27% hemolysis, results that were similar to the ones reported in the case of the isolates of high pathogenic potential. In our opinion, the hemolytic activity could be considered as an inherent characteristic of the amoeba and is induced at 37 °C, the reason why it is only detectable in the thermotolerant isolates. If this activity is important in the course of the infection and represents an advantage to the amoeba when it parasitizes a host requires more studies. Regarding the ADP production, it was not possible to detect the generation of this agonist by the amoebae employed. Previous studies using clinical isolates of Acanthamoeba (Mattana et al., 2001, 2002) reported that these amoebae possess the ability to secrete this molecule and, therefore, induce platelet aggregation; however, it has also been demonstrated that the expression of ecto-ATPases is lower in amoebae isolated from environmental samples, and that their activity is divalent cation-dependent (Sissons et al., 2004). The method of detection employed in this work requires a minimum concentration of 5 mM ADP, so we suggest a modification of this assay to increase sensitivity and determine if these amoebae are able or not of producing this agonist.
Sponsorships This work has been supported by Vicerrectoría de Investigación from University of Costa Rica (project 803-B7-117). CRediT authorship contribution statement Johan Alvarado-Ocampo: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing. Lissette Retana-Moreira: Writing - review & editing. Elizabeth Abrahams-Sandí: Conceptualization, Methodology, Investigation, Writing - original draft. Acknowledgements The authors want to thank the Investigation Center for Tropical Disease (CIET, by its initials in spanish), Clinical Analysis and Tumoral Chemosensitivity Laboratories and the Research Center in Hematology and Related Disorders (CIHATA, by its initials in spanish) for providing specialized equipment, research infrastructure and technical answers in some methodologies performed in this work. These centers belong to or are assigned to the Faculty of Microbiology, of the University of Costa Rica. We also want to thank Mr. Dennis Camareno-Carrillo for the technical assistance. References Awwad, S.T., Heilman, M., Hogan, R.N., Parmar, D.N., Petroll, W.M., McCulley, J.P., Cavanagh, H.D., 2007. Severe reactive ischemic posterior segment inflammation in Acanthamoeba keratitis: a new potentially blinding syndrome. Ophthalmol. Times 114, 313–320. Born, G.V., Cross, M.J., 1963. The aggregation of blood platelets. J. Physiol. 168, 178–195. Castro-Artavia, E., 2015. Determinación de características asociadas al potential patogénico de aislamientos de Acanthamoeba, obtenidos a partir de muestras de equipos de la Universidad de Costa Rica. Final graduation work to opt for the degree of Bachelor in Microbiology and Clinical Chemistry. Universidad de Costa Rica, San José 74 pp. Castro-Artavia, E., Retana-Moreira, L., Lorenzo-Morales, J., Abrahams-Sandi, E., 2017. Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers. Mem. Inst. Oswaldo Cruz 112 (12), 817–821. Chávez-Munguía, B., Omaña-Molina, M., González-Lázaro, M., González-Robles, A., Bonilla, P., Martínez-Palomo, A., 2005. Ultrastructural study of encystation and excystation in Acanthamoeba castellanii. J. Eukaryot. Microbiol. 52, 153–158. Chávez-Munguía, B., Salazar-Villatoro, L., Omaña-Molina, M., Rodríguez-Monroy, M.A., Segovia-Gamboa, N., Martínez-Palomo, A., 2014. Naegleria fowleri: contact dependent secretion of electrodense granules (EDG). Exp. Parasitol. 142, 1–6. Feingold, J.M., Abraham, J., Bilgrami, S., Ngo, N., Visvesvara, G.S., Edwards, R.L., Tutschka, P.J., 1998. Acanthamoeba meningoencephalitis following autologous peripheral stem cell transplantation. Bone Marrow Tranplant 22, 297–300. Flores-Maldonado, C., González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Gallardo, J.M., González-Lázaro, M., Hernández-Ramírez, V.I., Talamás-Rohana, P., Lorenzo-Morales, J., Martínez-Palomo, A., 2017. Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition. Exp. Parasitol. 183, 69–75. González-Garza, M.T., Castro-Garza, J., Cruz-Vega, D.E., Vargas-Villareal, J., CarranzaRosales, P., Mata-Cárdenas, B.D., Siller-Campo, L., Said-Fernández, S., 2000. Entamoeba histolytica: diminution of erythrophagocytosis, phospholipase A2, and hemolytic activities is related to virulence impairment in long-term axenic cultures. Exp. Parasitol. 96, 116–119. González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Lorenzo-Morales, J., Martínez-Palomo, A., 2013. Acanthamoeba royreba: morphological features and in vitro cytopathic effect. Exp. Parasitol. 133, 369–375. Hraoui-Bloquet, S., Rima, M., Kouzayha, A., Hleihel, W., Sadek, R., Desfontis, J.C., Fajloun, Z., Accary, C., 2014. Effect of the Montivipera bornmuelleri snake venom on human blood: coagulation disorders and hemolytic activities. Open J. Hematol. 5 (4), 1–9. https://doi.org/10.13055/ojhmt_5_1_4.140609. Iqbal, J., Naeem, K., Siddiqui, R., Khan, N.A., 2014. In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells. Exp. Parasitol. 145, 78–83.
5. Conclusions It has been demonstrated that viable trophozoites isolated from environmental samples have hemolytic activity at 37 °C, in contrast to their excretion/secretion products and/or metabolites of the amoebae extracts, who failed to induce hemolysis. A contact-dependent mechanism is proposed as the main hemolytic factor, which may trigger or stimulate the secretion of hemolytic effectors capable of diffusing through the culture medium. Erythrophagocytosis was also observed. It was also described, the ability of some environmental isolates of Acanthamoeba to generate a double hemolytic halo pattern over blood agar. This finding raises the need to specifically determine the main hemolytic factor and to study the mechanism by which it acts, in order to explain this phenomenon in detail in further investigations. Finally, 5
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Noble, R.C., Vosti, K.L., 1971. Production of double zones of hemolysis by certain strains of hemolytic streptococci of groups A, B, C, and G on heart infusion agar. Appl. Microbiol. 22 (2), 171–176. Oddó, D., 2006. Infecciones por amebas de vida libre. Comentarios históricos, taxonomía y nomenclatura, protozoología y cuadros anátomo-clínicos. Rev. Chil. infectol. 23 (3), 200–214. Palme, C., Steger, B., Haas, G., Teucher, B., Bechrakis, N.E., 2017. Severe reactive ischemic posterior segment inflammation in Acanthamoeba keratitis. Case report of a patient with Sjögren’s syndrome. Spekt. Augenheilkd. 31 (1), 10–13. Panjwani, N., 2010. Pathogenesis of Acanthamoeba keratitis. Ocul. Surf. 8 (2), 70–79. Shibayama, M., Siva-Olivares, A., González, S., Tsutsumi, V., 2005. Role of erythrophagocytosis in free-living amoebae. @inproceedings. https://www. semanticscholar.org/paper/Role-of-Erythrophagocytosis-in-Free-Living-AmoebaeShibayama-Silva-Olivares/b2ac4c052292b1872c0e76fa242b640ca66f14e5. Silva-Caumo, K., Mariante-Monteiro, K., Rodrigues-Ott, T., Maschio, V., Wagner, G., Bunselmeyer-Ferreira, H., Brittes-Rott, M., 2014. Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga. Acta Trop. 140, 166–172. Sissons, J., Alsam, S., Jayasekera, S., Khan, N.A., 2004. Ecto-ATPases of clinical and nonclinical isolates of Acanthamoeba. Microb. Pathog. 37, 231–239. Vargas-Ramírez, D., 2016. Aislamiento y caracterización de Acanthamoeba a partir de unidades combinadas de emergencia ubicadas en laboratorios docentes y de investigación de la Universidad de Costa Rica. Final graduation work to opt for the degree of Bachelor in Microbiology and Clinical Chemistry. Universidad de Costa Rica, San José 84 pp.
Khan, N.A., Jarrol, E.L., Panjwani, N., Cao, Z., Paget, T.A., 2000. Proteases as markers for differentiation of pathogenic and non-pathogenic species of Acanthamoeba. J. Clin. Microbiol. 38, 2858–2861. Khan, N.A., 2006. Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol. Rev. 30 (4), 564–595. Lorenzo-Morales, J., Khan, N.A., Walochnik, J., 2015. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite 22, 10. Maciver, S.K., Asif, M., Simmen, M.W., Lorenzo-Morales, J., 2013. Systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. Eur. J. Protistol. 49 (2), 217–221. Mattana, A., Tozzi, M.G., Costa, M., Delogu, G., Fiori, P.L., Cappuccinelli, P., 2001. By releasing ADP, Acanthamoeba castellanii causes an increase in the cytosolic free calcium concentration and apoptosis in Wish cells. Infect. Immun. 69, 4134–4140. Mattana, A., Cappai, V., Alberti, L., Serra, C., Fiori, P.L., Cappuccinelli, P., 2002. ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines. Infect. Immun. 70, 4424–4432. Mattana, A., Alberti, L., Delogu, G., Fiori, P.L., Cappuccinelli, P., 2009. In vitro activity of Acanthamoeba castellanii on human platelets and erythrocytes. Infect. Immun. 77 (2), 733–738. Nacapunchai, D., Permmongkol, C., Sripochang, S., Sermsart, B., Suvajeejarum, T., 2005. Comparison of physiological, cytopathogenic and immunological properties between two environmental isolates of Acanthamoeba spp. Southeast Asian. J. Trop. Med. Publ. Health 36 (4), 1–4.
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In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets
T
Johan Alvarado-Ocampoa,b, Lissette Retana-Moreiraa,b, Elizabeth Abrahams-Sandía,b,∗ a b
University of Costa Rica, Faculty of Microbiology, Department of Parasitology, San Pedro, San José, Costa Rica Centro de Investigación en Enfermedades Tropicales, San Pedro, San José, Costa Rica
A R T I C LE I N FO
A B S T R A C T
Keywords: Acanthamoeba Hemolytic activity Platelet aggregation Pathogenicity factor
Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperaturedependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.
1. Introduction Free-living amoebae (FLA) of the genus Acanthamoeba are widely distributed microorganisms that can be isolated from natural or artificial water bodies, drainages, sediments, air conditioning filters, surgical instruments, contact lenses, dialysis units, nasal cavities and skin lesions (Khan, 2006). They are considered emerging parasites, due to the increase and difficult handling of cases. In addition, they are known as amphizoic microorganisms, due to their ability to inhabit both external environments and the interior of living organisms, acting as a facultative parasite (Oddó, 2006). Acanthamoeba has been implicated in the development of severe infectious conditions such as amoebic granulomatous encephalitis and amoebic keratitis (infection of the cornea). Through molecular techniques, it has been determined that most of the isolates from human and environmental infections belong to the T4 genotype (Maciver et al., 2013). Studies regarding the main pathogenicity factors associated with these microorganisms allow the understanding of the mechanisms of
∗
damage the amoebae employ during the infection process. In Acanthamoeba, these factors can be of indirect origin (ubiquity, biofilm formation, resistance to therapeutic drugs, and physiological tolerance to osmolarity and temperature), which do not correspond to mechanisms that are activated to inflict damage, but that facilitate the survival of the amoeba in the pathogenic process (Lorenzo-Morales et al., 2015). On the other hand, there are also direct factors involved, which are both dependent and independent of contact. Contact-dependent factors are based on the adhesion of the amoeba to the tissues of the host as the first and most important step in the pathogenesis process. Here, extracellular matrix binding proteins are involved. In addition, processes such as phagocytosis and the release of harmful products such as the MIP-133 protease are activated (LorenzoMorales et al., 2015; Panjwani, 2010). Among the independent contact factors are soluble secretion products such as serine proteases, cysteine proteases, and other hydrolytic enzymes. These generate cytopathic effects that lead to cell death, the degradation of the extracellular matrix and the plasma membrane, with the eventual invasion of the
Corresponding author. University of Costa Rica, Faculty of Microbiology, Department of Parasitology, San Pedro, San José, Costa Rica. E-mail address: [email protected] (E. Abrahams-Sandí).
https://doi.org/10.1016/j.exppara.2020.107842 Received 3 December 2019; Received in revised form 10 January 2020; Accepted 18 January 2020 Available online 21 January 2020 0014-4894/ © 2020 Elsevier Inc. All rights reserved.
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Germany) and collected. The ACM was employed immediately after each collection process.
parasite to adjacent areas, promoting the persistence and aggravation of the infection (Panjwani, 2010; Khan et al., 2000) The damage mechanisms described above may involve cells such as erythrocytes and platelets (Mattana et al., 2009). Thus, hemolysis and platelet aggregation are factors that are also associated with pathogenicity and may represent a source of food for FLA or a direct target to carry out a successful invasion and the permanence inside the host. In other protozoa such as Entamoeba histolytica, this hemolytic capacity has been demonstrated (Lorenzo-Morales et al., 2015). Regarding Acanthamoeba, there are few studies related to the effects of Acanthamoeba on hemodynamics (Mattana et al., 2009; Nacapunchai et al., 2005). The description of clinical cases suggests that thrombosis in the blood vessels of the retina, necrotizing vasculitis (Awwad et al., 2007; Palme et al.,2017), hemorrhages and thrombi in the vessels of the periventricular tissue and the cerebellum, as well as other microcirculatory disorders (Feingold et al., 1998), could develop during a keratitis and a cerebral amebiasis. However, the mechanisms that generate this phenomenon are not yet clear and require further studies. Due to the scarcity of information in this area, the aim of this study was to evaluate the capacity of damage to some environmental isolates of Acanthamoeba and its products over blood elements.
2.3. Acanthamoeba trophozoite crude extracts Trophozoites of Acanthamoeba were suspended in phosphate buffer (PBS) (Sigma-Aldrich, St. Louis, USA) and 3 mL of each sample was centrifuged for 5 min at 5000 rpm. The pellet was resuspended in 1 mL of PBS and sonicated (10 cycles of 2 min each cycle) in an Ultrasonic Homogenizer (Cole-Parmer® Instrument Co., Illinois, USA). Finally, the samples were centrifuged at 7000 rpm for 6 min and filtered through a 0.22 μM pore membrane and the supernatant was collected. The crude extracts were employed immediately after each filtration process. 2.4. Hemolytic activity of live trophozoite, ACM and crude extracts over a suspension of erythrocytes 2.4.1. Live trophozoites For this assay, the protocol described by Mattana et al. (2009) was employed, with some modifications. Briefly, two suspensions were prepared: 1) a suspension of human erythrocytes in PBS (6 × 106 cells/ mL), and 2) a suspension of amoebae in PBS (3 × 106 amoebae/mL). The same volume of each suspension was added to 1.5 mL tubes (Eppendorf®, Hamburg, Germany), mixed and incubated for 0, 60 and 120 min at 37 °C. After this time, the samples were centrifuged for 10 min at 3000 rpm and the absorbance was measured in an UV-1800 spectrophotometer (Shimadzu® Corporation, Tokyo, Japan) at 546 nM. The blank solution employed was a suspension of erythrocytes in PBS (3 × 106 cells/mL) and the positive control (100% hemolysis) was a suspension of erythrocytes in distilled water (3 × 106 cells/mL). The hemolysis percentage was calculated as follows:
2. Material and methods 2.1. Amoebae The environmental isolates were characterized by the “Laboratorio de Protozoología Médica” of the University of Costa Rica (Table 1) (Castro-Artavia, 2015; Vargas-Ramírez, 2016) They were maintained in 25 cm2 cell culture flasks (Becton Dickinson®, New Jersey, USA) with peptone 2% (m/V), yeast extract 0,1% (m/V) and glucose 1,8 (m/V) massive growth medium (PYG), supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 10 μg/mL gentamicin (Sigma-Aldrich®, St. Louis, USA). Also, the evaluation of the environmental strain of Acanthamoeba castellanii Neff was included.
Hemolysis (%) = [Abstube – Absblank / Abscontrol+] * 100 Each assay was performed in triplicate. 2.4.2. ACM and crude extracts The activity of ACM and the crude extracts of trophozoites over erythrocytes were evaluated following the methodology described by Hraoui-Bloquet et al. (2014) with some modifications. Briefly, 100, 150 and 175 μL of a 30% suspension (V/V) of human erythrocytes in sterile saline solution 0.85% (m/V) were placed in Nunc™ 96-well ELISA plates (Thermo Fisher Scientific®, Massachusetts, USA). Then, 100, 50 and 25 μL of crude extract and ACM obtained after 24 h and 96 h of incubation were added, to obtain a final volume of 200 μL. The incubations were performed at 4, 30 and 37 °C and the absorbance of each well was measured in a Synergy HT plate reader (BioTek® Instruments Inc., Vermont, USA) at 540 nM. A lysate of erythrocytes in distilled water was employed as the positive control (100% hemolysis) and the
2.2. Acanthamoeba conditioned medium (ACM) To obtain the Acanthamoeba conditioned medium, the methodology described by Iqbal et al. was employed (Iqbal et al., 2014), with some modifications. Briefly, the PYG medium was removed from the culture flasks (with 1 × 106 amoebae/mL) and replaced with 3 mL of RPMI1640 (Sigma-Aldrich®, St. Louis, USA) with glucose 2% (m/V) (Merck Group, Darmstadt, Germany). Then, different amoebae cultures were incubated for 24 and 96 h at 30 °C and 2 mL of the conditioned medium was collected at the end of these times. Once collected, the medium was promptly centrifuged for 7 min at 3000 rpm, filtered through a 0.22 μM Minisart® syringe membrane (Sartorius Stedim Biotech, Goettingen,
Table 1 Axenic environmental isolates employed in these assays and their main characteristics related to pathogenic potential. Isolate
CSU6 CSU7 CSU8 CSU14 CSU18 DU3 DU13 H6 CSU7a H7
Sampling site
CSU: eyewash head CSU: eyewash sprayer CSU: eyewash sprayer CSU: eyewash head CSU eyewash sprayer Dental unit 3, Triple function syringe Dental unit 17, Triple function syringe Emergency Room: work table CSU powder Hospital water sample
Genotype
T4 T4 T4 T4 T4 T4 T4 T4 T4 T5
Cytopatic effect
Mild None Moderate Moderate High Mild Mild High Mild High
Thermotolerance*
Osmotolerance**
37 °C
40 °C
0, 5 M mannitol
1,0 M mannitol
+ + + + + + + + + +
+ – + – – + + + + –
+ + + + + + + + + +
+ – + + + + + + + –
Thermotolerance: proliferation at a 37 °C and 40 °C. **Osmotolerance: proliferation at different manitol concentrations, 0.5 M and 1.0 M. CSU: Combination shower unit. 2
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negative control included a suspension of erythrocytes in RPMI-1640 glucose 2% (m/V) or PBS. The percentage of hemolysis was calculated using the following formula:
Table 2 Percentage of hemolysis of a suspension of erythrocytes obtained for ten environmental isolates of Acanthamoeba and the Neff strain, at two different time points.
Hemolysis (%) = [Abswell – Abscontrol- / Abscontrol+] * 100
Isolate
Each assay was performed in triplicate. 2.5. Erythrophagocytosis assay
Neff CSU6 CSU7 CSU8 CSU14 CSU18 DU3 DU13 H6 CSU7a H7
2.5.1. Co-incubation of amoebae and erythrocytes Suspensions of erythrocytes (2 × 103 cells/μL) and amoebae (1 × 103 amoebae/μL) were prepared and the same volume of each suspension (cell:amoebae in a ratio 2:1) were placed in 96-well plates, incubated for 10 min at room temperature and then incubated for 60 min at 37 °C in a multimodal reader microscope Cytation (BioTek® Instruments Inc., Vermont, USA). Photographs of the samples were captured every 30 s and then analyzed in search of adhesion, phagocytic or lytic activity.
Average hemolysis percentage (%) ± SD 60 min
120 min
32.0 ± 0.7 24.4 ± 2.0 7.7 ± 2.2 6.4 ± 0.7 5.1 ± 1.3 2.6 ± 0.7 16.7 ± 3.9 5.1 ± 1.5 6.4 ± 2.7 1.3 ± 0.0 3.8 ± 0.7
42.3 41.0 24.4 15.4 39.7 16.7 26.9 16.7 35.9 18.0 25.6
± ± ± ± ± ± ± ± ± ± ±
2.0 1.3 1.5 2.7 2.2 3.2 2.0 0.7 3.4 0.7 0.7
CSU7, CSU8, CSU14, CSU18, DU13, H7 and H6 presented hemolysis percentages lower than 8% during the first hour and rose over 15% at the 2-hrs incubation period, while Neff, DU3 and CSU6 registered percentages greater than 15% in both incubation periods. Moreover, 80% of the isolates were able to produce a visible hemolytic effect over blood agar plates at the 5th-6th day of incubation. In this case, two patterns of hemolysis were registered: double halo (CSU6, H6 y CSU18) and another pattern similar to bacterial beta hemolysis (Neff, H7, CSU7, CSU14, DU3 y DU13) (Fig. 1). CSU7a and CSU8 showed results similar to the negative control. For this assay, it is important to highlight that, at 24–48 h of incubation, the encystment of most of the trophozoites placed over the agar plates was observed:
2.6. Hemolytic activity of live trophozoites, ACM and trophozoite crude extracts over blood agar plates In this case, the methodology described by Hraoui-Bloquet et al. (2014) was employed, with some modifications. Briefly, for the ACM and the crude extracts of trophozoites, 40 μL were placed in 7 mMdiameter wells inside 5% blood agar plates. In the case of complete trophozoites, a suspension of amoebae (1 × 106 amoebae/mL) in 50 μL PBS was placed on the surface of 5% blood agar plates. These plates were incubated at room temperature and 37 °C, with daily observation for one week. Controls of the assay included either PBS or RPMI-1640 with glucose 2% (m/V). The hemolytic activity of each sample was evaluated in quadruplicate. 2.7. Platelet aggregation assay ACM of each isolate was obtained as described above, with the following modifications: 1) PBS supplemented with 20 mM HEPES medium (Gibco®, Thermo Fisher Scientific®, Massachusetts, USA) was employed as the medium, and 2) amoebae were incubated for 2, 4 and 8 h at 30 °C prior to harvesting. Then, the samples were filtered through a 30 kDa membrane filter (Merck Millipore®, Darmstadt, Germany) and the supernatants were collected. The turbidimetric method described by Born and Cross (1963) was employed, with modifications. A volume of 5 μL of ACM (per each incubation time) was added inside the assay tube that contained an aliquot of 500 μL of platelet-rich plasma (PRP) (250,000 platelets/μL), to evaluate the increase in the passage of light and then create a platelet aggregation curve for a minimum of 5 min. An aliquot of 5 μL of 10 μM adenosine triphosphate (ADP) was employed as the positive control and an equal amount of PBS with 20 mM Gibco® HEPES medium was employed as the negative control. Also, a 500-μL aliquot of PRP (250,000 platelets/μL) was employed as the 0% transmittance solution, defined in the 490 4 + 4 optical aggregometer (Chronolog® Corporation, Pennsylvania, USA). The 100% transmittance solution corresponded to an equal aliquot of platelet-poor plasma (PPP). This assay was performed in triplicate. 3. Results No evidence of hemolytic activity was observed in the case of ACM and crude extracts of trophozoites, regardless of the incubation temperature and the type of assay performed. However, when the assays were performed employing live trophozoites, there was a marked hemolytic activity only at 37 °C. Hemolytic activity over erythrocyte suspensions was demonstrated in all of the isolates (Table 2). CSU7a,
Fig. 1. Effect of the living Acanthamoeba trophozoites over blood agar plates, after 6–8 days incubation at 37 °C: a. CSU6 (double halo pattern), b. CSU7 (simple halo pattern), c. CSU7a (no hemolisis). 3
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Fig. 2. Interaction between Acanthamoeba and a suspension of erythrocytes, during an incubation at 37 °C using light microscopy. Images were recorded at 15 min (a-b) and 45 min (c-d). Thick arrows indicate the multiple adhesion (a-c) and thin arrows show the deformation of the erythrocyte (c-d). Circles indicate the partial engulfment (c-d). Bar = 35 μM.
the limiting factor in their studies, both research groups refer to the low number of isolates assayed (only one or two). The aims of this work were to explore the hemolytic activity of ten environmental isolates of Acanthamoeba over human red blood cells, to assess the capacity of platelet aggregation by trophozoites through the production of the ADP agonist and to evaluate the phenomenon of erythrophagocytosis. All of the isolates employed were characterized by molecularly and physiologically in previous studies (Castro-Artavia, 2015; Vargas. Ramírez, 2016; Castro-Artavia et al., 2017) The results using erythrocytes in suspension showed a contact-dependent mechanism of hemolysis in all of the isolates. This was observed only when live trophozoites were used and at an incubation temperature of 37 °C, recording maximum activity at 2 h p.i. None of the conditioned media showed hemolytic activity. The above differs from the information published by Mattana et al. (2009), which reported, in addition to the activity of the live trophozoites, contact-independent hemolysis in the case of a clinical isolate of A. castellani. The authors proved the activity of soluble products secreted by the amoeba over erythrocytes in suspension, an activity that was Ca2+- and glucosedependent. More studies are needed to show if this haemolytic mechanism is exclusive of the clinical isolates. In the blood agar assay, only 80% of the isolates showed hemolytic activity. This may be related to the difference in the availability of the red blood cell in the agar plates, which could limit the phagocytosis and digestion of the cell by the amoebae. Another important aspect to be highlighted is that the hemolysis in the agar plates was observed until the 5th-6th day of incubation and at a temperature of 37 °C. The timelapse coincides with the encystment-excystment of trophozoites, which suggests that the effect observed in the plates could be the result of the release of products with hemolytic activity during these processes and it is not the direct destruction of the erythrocyte by the amoebae. Two different patterns of hemolysis were observed over the agar plates: a simple- (62,5%) or a double-halo (37,5%). According to the literature, the double halo hemolytic pattern has not been reported previously for these free-living amoebae, but a similar effect has been described in Streptococcus spp. In bacteria, this phenomenon is given by a non-synchronous production of hemolysins (Noble and Vosti, 1971) The possibility that the pattern observed in Acanthamoeba is linked to asynchronous mechanisms of secretion and diffusion of metabolites has
however, at 120–144 h p.i. They started the excystment process. In the erythrophagocytosis assay, the capacity of the amoebae to adhere to the erythrocytes was demonstrated, with the induction of cytoplasmic projections and morphological changes over these cells (Fig. 2). Erythrophagocytosis was also observed (Fig. 3). Finally, none of the tested ACM showed platelet aggregation capacity after 5 min of interaction with the platelet-rich plasma. According to the platelet aggregation curve, 0% aggregation was obtained for all of the samples tested. 4. Discussion The activity of Acanthamoeba over human erythrocytes has been poorly studied. In 2005, Nacapunchai et al. reported that environmental isolates with high pathogenic potential showed more adhesion to the erythrocyte than those with low pathogenic potential while Mattana et al. (2009) reported that these cells and platelets could be potential targets during the course of an Acanthamoeba castellani infection. In both studies, it was suggested that cell lysis occurs due to contact-dependent and -independent mechanisms that require the presence of some factors of the plasma and/or calcium ions and glucose. As
Fig. 3. Light microscopy image of an Acanthamoeba trophozoite with phagocytized erythrocytes after a 30 min incubation at 37 °C. Magnification: 40X, Bar = 40 μM. 4
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it is suggested that the platelet aggregation signal was not detected by the technique employed in this work, maybe due to ADP production at insufficient concentrations (< 5 mM).
not been ruled out and could be a subject for future research. In the present work, the erythrophagocytosis by Acanthamoeba was demonstrated, which contrasts with the findings reported by Nacapunchai et al. (2005) who did not observe the ingestion of red blood cells by the environmental isolates. As shown in Fig. 2, there was a process of adhesion of the amoebae to the erythrocytes, as well as a deformation of the red blood cell. However, the phagocytic index of all of the Acanthamoeba isolates was low, like the results reported by Shibayama et al. (2005). According to these authors, erythrophagocytosis is not a relevant mechanism in the pathogenesis of Acanthamoeba. Until now, most of the experiments that employ Acanthamoeba indicate that the contact-dependent mechanisms are the ones involved in the pathogenesis of the infection (González et al., 2013; FloresMaldonado et al., 2017), and the hemolytic activity is not the exception. It is not clear which factors or what is the exact mechanism the amoeba employs to lyse the erythrocytes. However, in the case of Acanthamoeba, there are reports of the secretion of proteases, lipases and the presence of some structures that, in other protozoan parasites, are related to the lysis of the red blood cells. For example, in Entamoeba histolytica, the hemolytic capacity is related to the presence of Ca2+dependent cytoplasmic phospholipases (González-Garza et al., 2000), while Naegleria fowleri secrete dense granules (in a contact-dependent way) that are associated to proteolysis and damage to the extracellular matrix (Chávez-Munguía et al., 2014). Phospholipases, serine- and cysteine-proteases and dense granules located near the ostiole have been described in Acanthamoeba (Chávez-Munguía et al., 2005; Silva-Caumo et al., 2014), so their role in the hemolytic activity of the amoeba could be subject to research. Our results showed that there is not an association between the level of cytopathic effect in vitro and the hemolytic activity since the isolates with a cytopathic effect reported as “none” or “mild” over the cell monolayer reached percentages of 15–27% hemolysis, results that were similar to the ones reported in the case of the isolates of high pathogenic potential. In our opinion, the hemolytic activity could be considered as an inherent characteristic of the amoeba and is induced at 37 °C, the reason why it is only detectable in the thermotolerant isolates. If this activity is important in the course of the infection and represents an advantage to the amoeba when it parasitizes a host requires more studies. Regarding the ADP production, it was not possible to detect the generation of this agonist by the amoebae employed. Previous studies using clinical isolates of Acanthamoeba (Mattana et al., 2001, 2002) reported that these amoebae possess the ability to secrete this molecule and, therefore, induce platelet aggregation; however, it has also been demonstrated that the expression of ecto-ATPases is lower in amoebae isolated from environmental samples, and that their activity is divalent cation-dependent (Sissons et al., 2004). The method of detection employed in this work requires a minimum concentration of 5 mM ADP, so we suggest a modification of this assay to increase sensitivity and determine if these amoebae are able or not of producing this agonist.
Sponsorships This work has been supported by Vicerrectoría de Investigación from University of Costa Rica (project 803-B7-117). CRediT authorship contribution statement Johan Alvarado-Ocampo: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing. Lissette Retana-Moreira: Writing - review & editing. Elizabeth Abrahams-Sandí: Conceptualization, Methodology, Investigation, Writing - original draft. Acknowledgements The authors want to thank the Investigation Center for Tropical Disease (CIET, by its initials in spanish), Clinical Analysis and Tumoral Chemosensitivity Laboratories and the Research Center in Hematology and Related Disorders (CIHATA, by its initials in spanish) for providing specialized equipment, research infrastructure and technical answers in some methodologies performed in this work. These centers belong to or are assigned to the Faculty of Microbiology, of the University of Costa Rica. We also want to thank Mr. Dennis Camareno-Carrillo for the technical assistance. References Awwad, S.T., Heilman, M., Hogan, R.N., Parmar, D.N., Petroll, W.M., McCulley, J.P., Cavanagh, H.D., 2007. Severe reactive ischemic posterior segment inflammation in Acanthamoeba keratitis: a new potentially blinding syndrome. Ophthalmol. Times 114, 313–320. Born, G.V., Cross, M.J., 1963. The aggregation of blood platelets. J. Physiol. 168, 178–195. Castro-Artavia, E., 2015. Determinación de características asociadas al potential patogénico de aislamientos de Acanthamoeba, obtenidos a partir de muestras de equipos de la Universidad de Costa Rica. Final graduation work to opt for the degree of Bachelor in Microbiology and Clinical Chemistry. Universidad de Costa Rica, San José 74 pp. Castro-Artavia, E., Retana-Moreira, L., Lorenzo-Morales, J., Abrahams-Sandi, E., 2017. Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers. Mem. Inst. Oswaldo Cruz 112 (12), 817–821. Chávez-Munguía, B., Omaña-Molina, M., González-Lázaro, M., González-Robles, A., Bonilla, P., Martínez-Palomo, A., 2005. Ultrastructural study of encystation and excystation in Acanthamoeba castellanii. J. Eukaryot. Microbiol. 52, 153–158. Chávez-Munguía, B., Salazar-Villatoro, L., Omaña-Molina, M., Rodríguez-Monroy, M.A., Segovia-Gamboa, N., Martínez-Palomo, A., 2014. Naegleria fowleri: contact dependent secretion of electrodense granules (EDG). Exp. Parasitol. 142, 1–6. Feingold, J.M., Abraham, J., Bilgrami, S., Ngo, N., Visvesvara, G.S., Edwards, R.L., Tutschka, P.J., 1998. Acanthamoeba meningoencephalitis following autologous peripheral stem cell transplantation. Bone Marrow Tranplant 22, 297–300. Flores-Maldonado, C., González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Gallardo, J.M., González-Lázaro, M., Hernández-Ramírez, V.I., Talamás-Rohana, P., Lorenzo-Morales, J., Martínez-Palomo, A., 2017. Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition. Exp. Parasitol. 183, 69–75. González-Garza, M.T., Castro-Garza, J., Cruz-Vega, D.E., Vargas-Villareal, J., CarranzaRosales, P., Mata-Cárdenas, B.D., Siller-Campo, L., Said-Fernández, S., 2000. Entamoeba histolytica: diminution of erythrophagocytosis, phospholipase A2, and hemolytic activities is related to virulence impairment in long-term axenic cultures. Exp. Parasitol. 96, 116–119. González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Lorenzo-Morales, J., Martínez-Palomo, A., 2013. Acanthamoeba royreba: morphological features and in vitro cytopathic effect. Exp. Parasitol. 133, 369–375. Hraoui-Bloquet, S., Rima, M., Kouzayha, A., Hleihel, W., Sadek, R., Desfontis, J.C., Fajloun, Z., Accary, C., 2014. Effect of the Montivipera bornmuelleri snake venom on human blood: coagulation disorders and hemolytic activities. Open J. Hematol. 5 (4), 1–9. https://doi.org/10.13055/ojhmt_5_1_4.140609. Iqbal, J., Naeem, K., Siddiqui, R., Khan, N.A., 2014. In vitro inhibition of protease-activated receptors 1, 2 and 4 demonstrates that these receptors are not involved in an Acanthamoeba castellanii keratitis isolate-mediated disruption of the human brain microvascular endothelial cells. Exp. Parasitol. 145, 78–83.
5. Conclusions It has been demonstrated that viable trophozoites isolated from environmental samples have hemolytic activity at 37 °C, in contrast to their excretion/secretion products and/or metabolites of the amoebae extracts, who failed to induce hemolysis. A contact-dependent mechanism is proposed as the main hemolytic factor, which may trigger or stimulate the secretion of hemolytic effectors capable of diffusing through the culture medium. Erythrophagocytosis was also observed. It was also described, the ability of some environmental isolates of Acanthamoeba to generate a double hemolytic halo pattern over blood agar. This finding raises the need to specifically determine the main hemolytic factor and to study the mechanism by which it acts, in order to explain this phenomenon in detail in further investigations. Finally, 5
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Noble, R.C., Vosti, K.L., 1971. Production of double zones of hemolysis by certain strains of hemolytic streptococci of groups A, B, C, and G on heart infusion agar. Appl. Microbiol. 22 (2), 171–176. Oddó, D., 2006. Infecciones por amebas de vida libre. Comentarios históricos, taxonomía y nomenclatura, protozoología y cuadros anátomo-clínicos. Rev. Chil. infectol. 23 (3), 200–214. Palme, C., Steger, B., Haas, G., Teucher, B., Bechrakis, N.E., 2017. Severe reactive ischemic posterior segment inflammation in Acanthamoeba keratitis. Case report of a patient with Sjögren’s syndrome. Spekt. Augenheilkd. 31 (1), 10–13. Panjwani, N., 2010. Pathogenesis of Acanthamoeba keratitis. Ocul. Surf. 8 (2), 70–79. Shibayama, M., Siva-Olivares, A., González, S., Tsutsumi, V., 2005. Role of erythrophagocytosis in free-living amoebae. @inproceedings. https://www. semanticscholar.org/paper/Role-of-Erythrophagocytosis-in-Free-Living-AmoebaeShibayama-Silva-Olivares/b2ac4c052292b1872c0e76fa242b640ca66f14e5. Silva-Caumo, K., Mariante-Monteiro, K., Rodrigues-Ott, T., Maschio, V., Wagner, G., Bunselmeyer-Ferreira, H., Brittes-Rott, M., 2014. Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga. Acta Trop. 140, 166–172. Sissons, J., Alsam, S., Jayasekera, S., Khan, N.A., 2004. Ecto-ATPases of clinical and nonclinical isolates of Acanthamoeba. Microb. Pathog. 37, 231–239. Vargas-Ramírez, D., 2016. Aislamiento y caracterización de Acanthamoeba a partir de unidades combinadas de emergencia ubicadas en laboratorios docentes y de investigación de la Universidad de Costa Rica. Final graduation work to opt for the degree of Bachelor in Microbiology and Clinical Chemistry. Universidad de Costa Rica, San José 84 pp.
Khan, N.A., Jarrol, E.L., Panjwani, N., Cao, Z., Paget, T.A., 2000. Proteases as markers for differentiation of pathogenic and non-pathogenic species of Acanthamoeba. J. Clin. Microbiol. 38, 2858–2861. Khan, N.A., 2006. Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol. Rev. 30 (4), 564–595. Lorenzo-Morales, J., Khan, N.A., Walochnik, J., 2015. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite 22, 10. Maciver, S.K., Asif, M., Simmen, M.W., Lorenzo-Morales, J., 2013. Systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. Eur. J. Protistol. 49 (2), 217–221. Mattana, A., Tozzi, M.G., Costa, M., Delogu, G., Fiori, P.L., Cappuccinelli, P., 2001. By releasing ADP, Acanthamoeba castellanii causes an increase in the cytosolic free calcium concentration and apoptosis in Wish cells. Infect. Immun. 69, 4134–4140. Mattana, A., Cappai, V., Alberti, L., Serra, C., Fiori, P.L., Cappuccinelli, P., 2002. ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines. Infect. Immun. 70, 4424–4432. Mattana, A., Alberti, L., Delogu, G., Fiori, P.L., Cappuccinelli, P., 2009. In vitro activity of Acanthamoeba castellanii on human platelets and erythrocytes. Infect. Immun. 77 (2), 733–738. Nacapunchai, D., Permmongkol, C., Sripochang, S., Sermsart, B., Suvajeejarum, T., 2005. Comparison of physiological, cytopathogenic and immunological properties between two environmental isolates of Acanthamoeba spp. Southeast Asian. J. Trop. Med. Publ. Health 36 (4), 1–4.
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