Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior

Parasitology International 74 (2020) 102002

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Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior

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Daniel Coronado-Velázqueza, Angélica Silva-Olivaresa, Federico Castro-Muñozledob, Luis Fernando Lares-Jiménezc, Libia Zulema Rodríguez-Anayad, Mineko Shibayamaa, ⁎ Jesús Serrano-Lunab, a

Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico b Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico c Departamento de Ciencias Agronómicas y Veterinarias, Instituto Tecnológico de Sonora, Av. 5 de Febrero 818 Sur, C.P. 85000, Cd. Obregón, Sonora, Mexico d CONACYT-Instituto Tecnológico de Sonora, Av. 5 de Febrero 818 Sur, C.P. 85000, Cd. Obregón, Sonora, Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: Acanthamoeba mauritaniensis T4D Epithelial cells Cytopathic effect Tight junction proteins Proteases PMSF

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

1. Introduction Acanthamoeba spp. are free-living amoebae (FLA) and are ubiquitously distributed in nature; they are found in soil, water, dust, air conditioning filters, domestic tap water, contact lenses, and lens cases [1]. Acanthamoeba spp. can become opportunistic microorganisms in immunocompromised patients, causing skin lesions and granulomatous amoebic encephalitis (GAE). Additionally, these protozoa are able to cause amoebic keratitis (AK) in healthy people [2]. A. castellanii and Acanthamoeba polyphaga (two pathogenic species) have been isolated from patients suffering from keratitis and encephalitis. On the other hand, nonpathogenic Acanthamoeba mauritaniensis was isolated from soil in Morocco, and until now, it has not been described as capable of producing cellular damage [3]. Pathogenic Acanthamoeba species cause damage through various pathogenic mechanisms, such as adhesion via a

130 kDa mannose binding protein (MBP) [4] and phagocytosis via structures such as food cups (amebostomes) [5]; more recently, pore forming proteins, or “acanthaporins”, were found in Acanthamoeba culbertsoni [6]. The Acanthamoeba group of FLA can produce and secrete a variety of proteases that can participate in the damage of host tissues, mainly in the cornea [7]. The main proteolytic enzymes present in these organisms are from the serine protease family [8,9]; contact-dependent metalloproteases [10], elastases [11] and cysteine proteases [8] have also been described. Our group reported that in a model of amoebic keratitis, A. castellanii is capable of detaching surface cells of the epithelium, thereby weakening the intercellular junctions in the first stages of infection [12]. In recent years, there have been reports of the cytopathic effect of A. castellanii on the human cornea in the early stages of interaction; these reports describe similar effects to those occurring on the hamster cornea [13]. In 2009, Khan and coworkers found that A.

⁎ Corresponding author at: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico. E-mail address: [email protected] (J. Serrano-Luna).

https://doi.org/10.1016/j.parint.2019.102002 Received 25 July 2018; Received in revised form 8 October 2019; Accepted 21 October 2019 Available online 24 October 2019 1383-5769/ © 2019 Published by Elsevier B.V.

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MEM at a cell:trophozoite ratio of 1:1 for 1, 3, 6, and 10 h. Later, the capacity for amoebic damage in the different cultures was evaluated by light microscopy (Nikon TMS, Tokyo, Japan).

castellanii degraded the tight junction (TJ) proteins ZO-1 and occludin in primary cell cultures of human brain microvascular endothelial cells (HBMEC), which constitute the blood brain barrier (BBB) [14]. TJs constitute the first barrier against microorganism invasion [15]. TJs are composed of the transmembrane proteins claudin (23 kDa) and occludin (65 kDa) [16]. The carboxyl termini of both occludin and claudin can associate with the Marvel-associated guanylate kinase (MAGUK) proteins ZO-1 (220 kDa), ZO-2 (160 kDa) and ZO-3 (130 kDa) [16–18]. Our group found that the environmental isolate A. mauritaniensis is able to damage rabbit corneal epithelial RCE1(5 T5) cells and MDCK cells in vitro. In the present study, we showed that this amoeba possesses serine, cysteine and metalloproteases in total crude extracts (TCE) and in conditioned medium (CM), with serine proteases being the main type of protease. Furthermore, A. mauritaniensis trophozoites were able to induce a decrease in MDCK cell transepithelial electrical resistance (TEER) by delocalizing and degrading the TJ proteins claudin-1, occludin and ZO-1 through the action of serine proteases.

2.4. Preparation of A. mauritaniensis T4D total crude extracts and conditioned medium

2. Materials and methods

Trophozoites were detached from the surface of the culture flasks by chilling the flasks in an ice bath, centrifuging at 466 ×g for 5 min, and washing twice with PBS (pH 7.4). Total crude extracts (TCE) were prepared by lysing 20 × 106 trophozoites with 10 freeze–thaw cycles. Protein content was quantified by the Bradford method [24]. To obtain conditioned medium (CM), we started with amoebic cultures (20 × 106 trophozoites) in the logarithmic growth phase (48 h) as reported by Serrano-Luna et al, (2006) [25]. CM was collected only after determining the viability of trophozoites using the trypan blue exclusion technique. The viability of the trophozoites was 98% (data not shown). The entire procedure was performed at 4 °C. The samples were stored at −80 °C until use [25].

2.1. Amoebic and cell cultures

2.5. Protease inhibitors for proteolytic assays

A. mauritaniensis (ATCC #50253) trophozoites were isolated from the soil [3], and this amoeba was kindly donated by Dr. Govinda Visvesvara (Centers for Disease Control and Prevention, Atlanta, United States). A. castellanii (isolated from a case of amoebic keratitis) was kindly provided by Dr. Simon Kilvington (Public Health Laboratory, Bath, UK). The amoebae were cultured at room temperature in Chang's liquid medium supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, TX, USA) according to a modified technique [3,19]. Trophozoites were harvested in logarithmic growth phase (48–72 h). Madin-Darby canine kidney (MDCK) cells were cultured in minimal essential medium (MEM) (Gibco, NY, USA) supplemented with 20% FBS in an atmosphere of 5% CO2 and at 37 °C. The rabbit corneal epithelial cell line RCE1(5 T5) [20] was kindly provided by Dr. Federico Castro-Muñozledo from the Department of Cell Biology, CINVESTAV-IPN and was cultured in a (3,1) DMEM/F12-Ham nutrient mixture supplemented with 5% FBS [21].

For the characterization of A. mauritaniensis T4D proteolytic patterns, different protease inhibitors were used. To inhibit cysteine proteases, 10 mM p-hydroximercuribenzoic acid (PHMB) (Sigma-Aldrich, MO, USA), 5 mM N-ethylmaleimide (NEM) (Sigma-Aldrich), and 10 μM trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64) (Roche, Basel, Switzerland) were used. To inhibit serine proteases, 5 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich) and 6 μM aprotinin (Sigma-Aldrich) were used. To inhibit metalloproteases, 2 mM ethylenediamine-tetraacetic acid (EDTA) (J.T. Baker, PA, USA) was used. We coincubated the samples with the protease inhibitors for 1 h; zymography and quantification of the proteolytic activity were performed using Azocoll (Millipore, MA, USA). 2.6. Azocoll quantitative assays Two milligrams of Azocoll was added to Eppendorf tubes (1.5 ml) with 250 μg of TCE or 200 μl of CM (from 20 × 106 trophozoites), and the final volume was adjusted to 500 μl by the addition of the following activation buffers: 100 mM acetic acid (Sigma-Aldrich) (pH 3.0), 100 mM sodium acetate (Sigma-Aldrich) (pH 5.0), 100 mM Tris–OH (Sigma-Aldrich) (pH 7.0), and 100 mM glycine (AMRESCO, OH, USA) (pH 9.0). All buffers contained 2 mM CaCl2 (J.T. Baker). TCE and CM were incubated overnight with shaking at 37 °C in triplicate. The reactions were stopped with 500 μl of 10% trichloroacetic acid (TCA) (Sigma-Aldrich), and the samples were centrifuged at 4600 ×g for 15 min. Supernatants were collected, and their absorbances were read with a spectrophotometer at 520 nm to determine the Azocoll activities as reported by Serrano-Luna et al, (2006) [25]. Protease activities were reported as milliunits per milligram; this unit of measure is equivalent to the amount of substrate degraded in 1 min per milligram of TCE or per milliliter of CM [9]. TCE and CM were preincubated with protease inhibitors (at the concentrations described above) at room temperature for 1 h before incubation with the substrate. The data were plotted using GraphPad Prism software (La Jolla, CA, USA). The bars represent the means ± SD of at least three independent experiments. The p values were calculated using two-way ANOVA.

2.2. A. mauritaniensis genotyping DNA of Acanthamoeba axenic culture was extracted using the DNeasy extraction kit (QIAGEN, Inc., CA, USA), followed by measurement of the DNA concentration with a NanoDrop 2000c spectrophotometer (Thermo Scientific, MA, USA). The Acanthamoeba primer set was JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′-TCTC ACAAGCTGCTAGGGAGTCA-3′ [22]. PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and sequenced in both directions. Once cyclic sequencing was obtained, the sample was purified using Centri-Sep™ Columns (Life Technologies, CA, USA). Once the sample was clean, the sequencing process began. The sequencing was carried out in an ABI PRISM® 310 Genetic Analyzer (Foster City, CA, USA). The phylogenetic tree was made with Maximum likelihood in the MEGAX program, using the “complete” Rns sequences indicated by Fuerst et al, (2015) [23], as representative for each genotype and compering the allelic sequence differences of the DF3 region. Of these tree, T99 was eliminated and types T15, T21 and T22 were added. 2.3. Cytopathic effect assays

2.7. Detection of A. mauritaniensis T4D proteolytic activity in SDS-PAGE copolymerized with gelatin substrate

For the interaction of A. mauritaniensis T4D and A. castellanii (data not shown) trophozoites with RCE1(5 T5) cells, we used serum-free DMEM at 37 °C and a cell:trophozoite ratio of 1:1 for 1, 3, and 6 h. The assessment of the interaction of A. mauritaniensis T4D and A. castellanii trophozoites with MDCK cells was performed at 37 °C in serum-free

Protease activity was analyzed after separating A. mauritaniensis T4D TCE and CM by 12% SDS-PAGE on a gel containing 0.1% porcine skin gelatin as a copolymerized substrate. Protein concentrations of 30 μg of TCE and 40 μl of CM were loaded per well. Electrophoresis was 2

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experiments. The p values were calculated using two-way ANOVA.

performed at 100 V at 4 °C for 3 h; gels were then washed twice with a 2.5% Triton X-100 solution for 15 min with shaking (Sigma-Aldrich) [25]. The zymogram gels were prepared in duplicate. To characterize the types of proteases, we preincubated TCE or CM with the protease inhibitors (described above) for 1 h, and electrophoresis was then performed as previously described.

2.10. Protease inhibitors for MDCK cell lysates For the preservation of MDCK cell lysates, we used protease inhibitors composed of the following: to inhibit cysteine proteases, 10 mM PHMB (Sigma-Aldrich), 5 mM NEM (Sigma-Aldrich), and 10 μM E-64 (Roche); to inhibit serine proteases, 5 mM PMSF (Sigma-Aldrich) and 6 μM aprotinin (Sigma-Aldrich); and to inhibit metalloproteases, 2 mM EDTA (J.T. Baker).

2.8. Delocalization of tight junction proteins induced by A. mauritaniensis T4D trophozoites MDCK monolayers were grown to 98% confluence on coverslips pretreated with 3% silane (Sigma-Aldrich), and 5 × 105 cells were coincubated with A. mauritaniensis T4D trophozoites at a 1:1 ratio. After 1, 3, 6 and 10 h of coincubation, MDCK cells were washed twice in cold PBS (4 °C) to detach the amoebae, and the cells were then fixed with 2% paraformaldehyde in PBS for 20 min at room temperature. Samples were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 15 min at room temperature and washed three times with cold PBS. Coverslips were blocked with 1% albumin for 1 h at room temperature. Cells were labeled with a polyclonal rabbit anti-occludin antibody (1:25), polyclonal rabbit anti-claudin-1 antibody (1:25) and polyclonal rabbit anti-ZO-1 antibody (1:25) (Zymed, CA, USA). Amoebae were labeled with polyclonal mouse antibodies against A. mauritaniensis T4D (1:50). The samples were washed 3 times with PBS and incubated with the following secondary antibodies: FITC-goat anti-rabbit (1:50) (Invitrogen, CA, USA), and CY5-goat anti-mouse (trophozoites) (1:50) (Santa Cruz Biotechnology, TX, USA) at 37 °C for 1 h. Finally, cell nuclei were stained with 0.001% propidium iodide (Sigma-Aldrich). The samples were stored in the dark at 4 °C until use. The samples were analyzed with a LEICA confocal microscope (TCS SP8) (Leica Microsystems, Glattbrugg, Switzerland). The fluorescence was quantified and reported as the corrected total cell fluorescence (CTCF). Briefly, an outline was drawn around each cell; the area and mean fluorescence were measured with ImageJ software (https://rsb.info.nih. gov/nih-image). The corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = integrated density – (area of selected cell × mean fluorescence of background readings) [26]. The quantification of the fluorescence signal was plotted with GraphPad Prism software (La Jolla, CA, USA). The data represent the means ± SD of at least three independent experiments. The p values were calculated using two-way ANOVA.

2.11. Analysis of MDCK TJ protein degradation by A. mauritaniensis T4D and analysis of protection against degradation using the serine protease inhibitor PMSF For the evaluation of TJ protein degradation, MDCK cells were incubated with A. mauritaniensis trophozoites in serum-free MEM. Subsequently, the amoebae were detached from the epithelial monolayers with cold PBS. On the other hand, to evaluate the protective effect toward TJ proteins, MDCK cells were incubated with trophozoites of A. mauritaniensis previously incubated for 1 h with 5 mM PMSF, washed twice with sterile PBS and coincubated in serum-free MEM for 10 h. Subsequently, the amoebae were detached from the epithelial monolayers with cold PBS. The MDCK cell extracts were obtained using lysis buffer [150 mM NaCl, 1% Triton X-100 and 50 mM Trizma base (pH 8.4)] (Sigma-Aldrich) with protease inhibitors (concentrations previously described for MDCK lysates) and stored at −80 °C until use. Protein samples were processed in sample buffer (Laemmli 5×) containing 5% β-mercaptoethanol (Sigma-Aldrich). Protein was loaded at 30 μg per well. The samples were resolved using 7.5%, 10%, 12% and 15% SDS-PAGE. Electrophoresis was performed at 4 °C at 100 V for 3 h, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked at room temperature with 7% skim milk dissolved in PBS and incubated overnight at 4 °C [27]. For the detection of TJ proteins, the PVDF membranes were incubated with polyclonal rabbit anti-occludin (1:200), polyclonal rabbit anti-claudin-1 (1:200), polyclonal rabbit anti-ZO-1 (1:300) and polyclonal mouse anti-actin (1:500) antibodies (Invitrogen) for 1 h at 37 °C. The secondary antibodies were used as follows: goat anti-rabbitIgG peroxidase-conjugated (1:500) and goat anti-mouse-IgG peroxidase-conjugated (1:500) (Santa Cruz Biotechnology). The membranes were washed six times with 0.005% PBS-Tween and developed with a luminol kit reagent (Santa Cruz Biotechnology) using Kodak photographic film. The densitometry analysis was performed with ImageJ software (https://rsb.info.nih.gov/nih-image), the results were expressed as the relative optical density (ROD), and the data were plotted using GraphPad Prism software (La Jolla, CA, USA). The bars represent the means ± SD of at least three independent experiments. The p values were calculated using two-way ANOVA.

2.9. Transepithelial electrical resistance (TEER) in MDCK cells after coincubation with A. mauritaniensis T4D The TEER values of MDCK cell cultures were measured directly using an epithelial voltohmmeter (EVOM) (World Precision Instruments, FL, USA). Briefly, 6 × 105 MDCK cells were coincubated at a 1:1 ratio with A. mauritaniensis T4D trophozoites in serum-free MEM, seeded in Millicell inserts with an 8 μm pore filter and a diameter of 1.12 cm2 (Millipore, USA) and plated into 12 well culture plates (Corning-Costar, NY, USA) containing the MDCK cell monolayers. The resistance was registered in ohms (Ω cm−2). When the resistance was stable (approximately 400 Ω cm−2), the MDCK monolayers were coincubated with A. mauritaniensis T4D trophozoites in serum-free MEM. To evaluate the protection of the monolayer, we preincubated the amoebae for 1 h with 5 mM PMSF and then coincubated them with the MDCK cells. For the negative control, we measured the TEER in an MDCK monolayer without amoebae. As a positive control, 25 mM NaOH was added to MDCK cells. The TEER was quantified for periods of 0, 5, 15, 30, 40 and 60 min. The resistance data were obtained from three independent experiments. The results were compared with those of the experimental controls (cells incubated either without amoebae or with PMSF alone) and were reported as percentages of the control results. The data were plotted using GraphPad Prism software (La Jolla, CA, USA). The data represent the means ± SD of at least three independent

3. Results 3.1. Acanthamoeba mauritaniensis genotyping Molecular identification of the amoebic strain was carried out by sequencing the PCR products generated with JDP1 and JDP2 primers in the 18S rDNA of Acanthamoeba [22]. The sequence corresponded to a Acanthamoeba mauritaniensis genotype T4D (A.mauritaniensis T4D) of the 18S rRNA Gene Typing System of Acanthamoeba GenBank accession number MN096650 (Fig. 1). 3.2. Cytopathic effect of A. mauritaniensis T4D trophozoites on RCE1(5 T5) and MDCK cell cultures Because A. castellanii is capable of causing amoebic keratitis in humans, we decided to evaluate whether A. mauritaniensis T4D could 3

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Fig. 1. Phylogenetic tree of Acanthamoeba strains based on 18S rDNA sequences.

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Fig. 2. Damage to RCE1(5 T5) and MDCK cells produced by A. mauritaniensis T4D trophozoites. A) RCE1(5 T5) cells without amoebae at 3 h. Corneal cells at 1 h of coincubation, showing trophozoites among and between the cells (arrowheads), and cellular damage was observed (arrows). At 3 h of coincubation, the loss of intercellular junctions was observed (asterisks), and amoebae appeared near the intercellular junctions (arrowheads). Bar = 25 μm. B) Incubation of A. castellanii and A. mauritaniensis T4D with MDCK cells at 1, 3, 6 and 10 h. MDCK cells without amoebae show an intact monolayer (1–10 h); MDCK cells coincubated with A. castellanii (arrowheads) show severe damage (asterisks) to the monolayer at 1 h that increase until 10 h of coincubation. MDCK cells coincubated with A. mauritaniensis T4D (arrowheads) show lytic areas (asterisks) at 1 h that increase until 10 h of coculture. Bar = 20 μm.

detaching the RCE1 monolayer. Because these two strains of Acanthamoeba induced substantial damage with detachment of the monolayer at early incubation times, we decided to evaluate the cytopathic effect in MDCK cells after 1, 3, 6 and 10 h at a 1:1 ratio (Fig. 2B). Our results revealed that MDCK cells without amoebae maintained normal morphology and viability during the entire assay duration (1−10h) (Fig. 2B). When we coincubated A. mauritaniensis T4D with MDCK cells, we observed that the amoebae were capable of damaging the MDCK monolayer by separating and lysing the cells after the first hour of coincubation, and the damage was more evident at 10 h of incubation (Fig. 2B). Moreover, the damage caused by A. mauritaniensis T4D was similar to the cytopathic effect produced by A. castellanii (Fig. 2B). Our results showed that A. mauritaniensis T4D and A. castellanii induced similar cytopathic effects in MDCK monolayers. Considering all these results, we decided to evaluate the role of proteases that could be

damage corneal cells. Therefore, we coincubated the amoebae with a cell line of keratinocytes from rabbit corneal epithelium, namely, RCE1(5 T5) (Fig. 2A). We incubated A. mauritaniensis T4D and A. castellanii with RCE1(5 T5) cultures at a 1:1 ratio of cells:amoebae in serum-free DMEM for 3 h. After the first hour of coincubation with A. mauritaniensis T4D, amoebae were observed among the intercellular junctions, and few areas of cell damage were observed; at 3 h postincubation, interestingly, we observed amoebae among and over the cells and in a few areas where the cellular junctions had been lost (Fig. 2A). At 6 h postinfection, the RCE1 corneal monolayer was detached from the culture plate (data not shown). Additionally, when we coincubated the cells with A. castellanii, the RCE1 monolayers displayed a behavior similar to that resulting from coincubation with A. mauritaniensis T4D (data not shown). From these data, we can conclude that these trophozoites can damage corneal cells and are capable of 5

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Fig. 3. Protease activities in TCE and CM of A. mauritaniensis T4D incubated with protease inhibitors at 37 °C and at different pH values (3, 5, 7 and 9) using Azocoll. TCE (A-C); A) incubated with 6 μM aprotinin (gray bars) and 5 mM PMSF (white bars), B) incubated with 10 μM E-64 (gray bars) and 5 mM NEM (white bars), and C) incubated with 2 mM EDTA (gray bars). CM (D-F); D) incubated with 6 μM aprotinin (gray bars) and 5 mM PMSF (white bars), E) incubated with 10 μM E-64 (gray bars) and 5 mM NEM (white bars), and F) incubated with 2 mM EDTA (gray bars). Controls of TCE and CM without protease inhibitors were used at all pH values mentioned above (black bars). The data represent the means ± SD of a least three independent experiments. The p values were calculated with two-way ANOVA (**p < .001, *p < .05).

pH 7 and 37 °C, as was reported for A. castellanii [4,11,25,28]. When we incubated TCE with the serine protease inhibitors aprotinin and PMSF, we observed that PMSF inhibited all the proteolytic bands (bands of 141, 105, 71.2, 53 and 40 kDa) (Fig. 4A); meanwhile, when we incubated TCE with aprotinin, a partial inhibition in the band intensities was observed (bands of 141, 53 kDa) (Fig. 4A). Additionally, when the samples were incubated with the cysteine protease inhibitor E-64 and the metalloprotease inhibitor EDTA, we observed a partial inhibition of the protease pattern (Fig. 4A) compared with the control. Moreover, we analyzed the proteolytic activities of the CM of A. mauritaniensis T4D at pH 7 and 37 °C (Fig. 4B). We observed a similar pattern in the CM: PMSF showed a better inhibition of the protease activities (bands of 164, 100, 70.5, 40 and 29 kDa), aprotinin induced less inhibition (bands of 100 and 29 kDa) (Fig. 4B), and E-64 and EDTA inhibited the 30 kDa proteolytic band (Fig. 4B). From these results, we suggest that the main protease activity in both TCE and CM is mediated by serine proteases and, to a lesser extent, by cysteine and metalloproteases (Table 1) [8,25].

involved in the cellular damage produced by A. mauritaniensis T4D. 3.3. Quantification of the proteolytic activity in TCE and CM of A. mauritaniensis T4D To determine the types of proteases present in A. mauritaniensis T4D that could participate in epithelial damage, we performed a partial biochemical characterization of the amoebic proteases in both TCE and CM by quantifying the proteolytic activity using Azocoll at pH values of 3, 5, 7 and 9 and incubating at 37 °C. We observed proteolytic activities at pH values of 3, 5, 7 and 9 for TCE (Fig. 3A-C) and CM (Fig. 3D-F). Moreover, we found that the serine protease inhibitors aprotinin and PMSF mainly inhibited the proteolytic activities of TCE and CM at pH values of 3, 5, 7 and 9 (Fig. 3A, D), and this inhibition was more evident in CM at pH values of 5, 7 and 9 with PMSF (Fig. 3D); we also observed inhibition with the cysteine protease inhibitors E-64 and NEM at the same pH conditions in TCE and CM (Fig. 3B, E). When we incubated the samples with EDTA (metalloprotease inhibitor), we observed less inhibition of protease activity than we observed with the serine and cysteine protease inhibitors in TCE at pH values of 5, 7 and 9 (Fig. 3C). In CM, the activity was inhibited at pH values of 5 and 7 (Fig. 3F). From these results, we confirmed that the main protease activity of A. mauritaniensis T4D was mediated by serine proteases and, to a lesser extent, by cysteine and metalloproteases in both TCE and CM (*p < .05, **p < .001).

3.5. MDCK TJ protein integrity after coincubation with A. mauritaniensis T4D To evaluate the effect of coincubating A. mauritaniensis T4D trophozoites with epithelial cells on MDCK cell TJ proteins, we analyzed the ZO-1, claudin-1 and occludin proteins using specific antibodies (Fig. 5A). We observed that the delocalization of ZO-1, claudin-1 and occludin induced by A. mauritaniensis T4D was evident after 1 h of coculture, and a substantial decrease in TJ protein labeling was observed until 10 h (Fig. 5A). The delocalization of TJ proteins by A. mauritaniensis T4D was compared with the effect induced by A. castellanii, where we observed a rearrangement of TJ proteins beginning at 1 h and lasting until 10 h of coincubation (data not shown). The labeling of TJ

3.4. Effect of protease inhibitors on the protease activity in TCE and CM of A. mauritaniensis T4D Once we quantified the proteolytic activity of A. mauritaniensis T4D, we analyzed the proteolytic profile using gelatin zymography for both TCE (Fig. 4A) and CM (Fig. 4B) incubated with protease inhibitors at 6

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Fig. 4. Zymograms of TCE and CM of A. mauritaniensis T4D incubated with protease inhibitors at pH 7 and at 37 °C. A) TCE and B) CM. Control without inhibitors (lane 1), incubation with 6 μM aprotinin (lane 2), 5 mM PMSF (lane 3), 10 μM E-64 (lane 4), or 2 mM EDTA (lane 5). Bands of 141, 105, 71.2, 53 and 40 kDa were observed in TCE. Bands of 164, 100, 70.5, 40 and 29 kDa were observed in CM. The zymograms were carried out in duplicate.

not degraded at any incubation time. The densitometry analysis demonstrated the degradation of TJ proteins in a time-dependent manner (Fig. 6C). From these results, we can conclude that A. mauritaniensis (T4D) was able to increase cellular permeability and induce the degradation of TJ proteins in a time-dependent manner (*p < .05).

Table 1 Summary of the proteolytic bands produced by the environmental strain A. mauritaniensis genotype T4D incubated at 37 °C and pH 7. Serine proteases (kDa)

Total crude extracts

Conditioned medium

Cysteine proteases (kDa)

141 105 71 40 18 162 70.5

100 69

40

29

Metallo-proteases (kDa)

100 70 40

3.6. The serine protease inhibitor PMSF protects MDCK cells from A. mauritaniensis T4D cytopathic damage and the alteration of TJ proteins To analyze whether the damage to the MDCK monolayer and the alteration in TJ proteins were induced by amoebic serine proteases, we preincubated the trophozoites with PMSF and then coincubated them with MDCK cells. Our results showed that PMSF inhibited the cytopathic effect in the MDCK monolayer during the duration of the assay (Fig. 7A). Additionally, we analyzed the TEER of MDCK cells cocultured with trophozoites preincubated with PMSF (Fig. 7B). As controls, we used MDCK cells cultured without trophozoites and MDCK cells incubated only with the inhibitor PMSF. As a positive control, we used MDCK monolayer incubated with 25 mM NaOH and MDCK cells coincubated with A. mauritaniensis T4D for 60 min. When we coincubated the cells with amoebae treated with PMSF, the TEER values were maintained during all evaluated times compared with the TEER values of the controls (Fig. 7B). Finally, we decided to analyze whether PMSF was able to protect MDCK cell TJ proteins against degradation by A. mauritaniensis T4D using Western blot assays (Fig. 7C). The results showed that PMSF protected the proteins ZO-1, claudin-1 and occludin against degradation during the evaluated times compared with the degradation observed in the assays performed with trophozoites without the inhibitor, which showed the degradation of the evaluated TJ proteins at 10 h of coculture (Fig. 7C). As a loading control, we used actin, which was not degraded during the entire duration of the assay. The densitometry analysis showed the protection of TJ proteins against degradation compared with the degradation observed in the controls of amoebae without PMSF (Fig. 7D). These results strongly suggest that A. mauritaniensis T4D serine proteases participate in cellular damage that consequently alters epithelial permeability and induces the degradation of intercellular junction proteins in a time-dependent manner

~30

proteins was quantified by measuring fluorescence intensity; the corrected total cell fluorescence (CTCF) was determined using ImageJ software (Fig. 5B). The CTCF showed that the delocalization induced by this amoeba was significant beginning at 1 h of coculture for ZO-1, claudin-1 and occludin (Fig. 5B). From these results, we can conclude that A. mauritaniensis T4D was able to delocalize the TJ proteins in a time-dependent manner (*p < .05, **p < .001). To analyze the damage induced by A. mauritaniensis T4D trophozoites on MDCK cell TJ proteins, we performed TEER measurements in MDCK cells (Fig. 6A). We observed a decrease of 5% in the TEER values beginning at 5 min of coculture with trophozoites and a decrease of 26% at 60 min compared with the TEER value of the control (MDCK cells without amoebae) (Fig. 6A). As a positive control for damage, 25 mM NaOH was used; we observed a 30% decrease in the TEER value starting at 5 min and a continued decrease in the TEER value to 95% at 60 min (Fig. 6A). To determine the possible degradation of TJ proteins by A. mauritaniensis T4D coincubated with MDCK cells at durations of 1, 3, 6 and 10 h, we performed Western blot assays (Fig. 6B). We observed that amoebae induced the degradation of ZO-1, claudin-1 and occludin in a timedependent manner. ZO-1 degradation was more evident at 6 and 10 h of coincubation; for claudin-1 and occludin, the degradation was significant from 3 to 10 h. As a loading control, we used actin, which was 7

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Fig. 5. MDCK TJ proteins were delocalized by A. mauritaniensis T4D trophozoites. A) A. mauritaniensis T4D trophozoites were cocultured with MDCK; ZO-1, claudin-1 and occludin were analyzed at 1, 3, 6 and 10 h. ZO-1, claudin-1 and occludin were immunolabeled with FITC coupled goat anti-rabbit antibodies (green), cell nuclei were labeled with propidium iodide (red), and the trophozoites were labeled with CY5 coupled goat anti-mouse antibodies (blue). Arrows show the loss of TJ immunolabeling beginning at 1 h until its disappearance at 10 h of coincubation. Bar = 50 μm. B) The fluorescence signal was plotted as the corrected total cell fluorescence (CTCF). The data represent the means ± SD of three independent assays. The p values were calculated using two-way ANOVA (*p < .05; **p < .001).

Fig. 6. TEER measurement of MDCK monolayer and TJ protein degradation during coincubation with A. mauritaniensis T4D. A) MDCK TEER values during coincubation with A. mauritaniensis T4D trophozoites. MDCK without amoebae (-o-). MDCK incubated with 25 mM NaOH as a positive control of damage (-□-). MDCK coincubated with amoebae (-Δ-). The data represent the means ± SD of at least three independent experiments. The p values were calculated with two-way ANOVA (*p < .05). B) Analysis of the degradation of MDCK TJ proteins induced by amoebae. Western blot analysis of ZO-1, claudin-1 and occludin, showing the degradation of TJ proteins from 1 to 10 h of coincubation. Actin was used as a loading control. C) The relative optical density (ROD) was determined for the TJ proteins (gray bars) and normalized to the actin load control. The interactions were compared with the controls without amoebae (black bars). The data represent the means ± SD of at least three independent experiments. The p values were calculated with two-way ANOVA (*p < .05).

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Fig. 7. PMSF protects MDCK cells and their intercellular junctions from amoebic proteases. A) Cytopathic effect assay using MDCK monolayer. Control MDCK cells without amoebae at 10 h. MDCK cells coincubated with A. mauritaniensis T4D (arrowheads). MDCK cells incubated with 5 mM PMSF. A. mauritaniensis T4D (arrowheads) preincubated with 5 mM PMSF and coincubated with MDCK cells for 10 h. Bar = 25 μm. B) TEER of MDCK cells cocultured with A. mauritaniensis T4D. As a control, MDCK cells without amoebae (-o-). MDCK incubated with 5 mM PMSF (-Δ-). As a positive control of damage, MDCK cells incubated with 25 mM NaOH (-□-). MDCK cells coincubated with amoebae (-▼-). MDCK coincubated with trophozoites preincubated with 5 mM PMSF (-♦-). The data represent the means ± SD of a least three independent experiments. The p values were calculated with two-way ANOVA (*p < .05). C) Western blot analysis of the degradation of MDCK TJ proteins (ZO-1, claudin-1, and occludin). MDCK cells coincubated with A. mauritaniensis T4D trophozoites previously incubated with 5 mM PMSF for 1, 3, 6 and 10 h (lanes 1–4). MDCK cocultured with A. mauritaniensis T4D for 10 h (lane 5), and MDCK incubated with 5 mM PMSF for 10 h (lane 6). Actin was used as a loading control. D) The relative optical density (ROD) was determined for the TJ proteins and normalized to the actin loading control. The interactions (white bars) were compared with the controls with amoebae (light gray bars) and amoebae incubated with PMSF (dark gray bars). The data represent the means ± SD of a least three independent experiments. The p values were calculated using two-way ANOVA (*p < .05).

to the damage caused by the pathogenic strain A. castellanii. This phenomenon strongly suggested a pathogenic behavior; for this reason, we decided to genotype this strain. The results demonstrated after genotyping that this is an isolate of genotype T4D was capable of causing damage to MDCK cells. Based on the rRNA sequences, the Acanthamoeba genus is currently divided into T1 to T22 genotypes [33], with genotype T4 being the most common genotype in clinical cases [34]. To date, there is no reliable animal model to evaluate Acanthamoeba virulence; however, an experimental model using BALB/c mice has recently been reported, in which the authors showed the presence of A. culbertsoni in the mouse brain [35]. Therefore, we decided to evaluate the amoebic cytopathic effect using a cell line derived from a primary culture of rabbit corneal epithelial cells [RCE1(5 T5)] [20]; these cells were more suitable as target cells to analyze the damage produced by A. mauritaniensis T4D. In the present work, we demonstrated that A. mauritaniensis T4D trophozoites were able to induce cytopathic effect on RCE1(5 T5) cells, detaching them from the collagen substrate formed by 3 T3 fibroblasts in the cultures in a short period of time and thus making it difficult to evaluate the damage to RCE1(5 T5) cells induced by A. mauritaniensis T4D. Therefore, we suggest that A. mauritaniensis T4D has a similar capacity to damage corneal cells as the pathogenic

(*p < .05). 4. Discussion It is known that > 24 species of Acanthamoeba have been described using morphological criteria; some of these species, such as A. castellanii, A. polyphaga, A. culbertsoni and Acanthamoeba rhysodes are able to induce infections in humans [29,30]. In particular, A. castellanii has been reported as the causal agent of GAE and skin lesions in immunocompromised patients and to cause AK in healthy patients [1]. However, other Acanthamoeba species that were isolated from the environment have been used as nonpathogenic reference strains. The freeliving amoeba A. mauritaniensis was previously characterized as a nonvirulent amoeba, was isolated from soil in Morocco and was not reported to produce disease in humans [3]. It is known that A. mauritaniensis has a different riboprint and protein pattern compared with the virulent strain A. castellanii; moreover, A. mauritaniensis did not damage Vero cell cultures [3,31,32]. In our laboratory, we have a nonvirulent A. mauritaniensis strain donated by Dr. Govinda Visvesvara (Centers for Disease Control and Prevention, Atlanta, United States). However, this amoeba was able to induce damage to MDCK cells similar 9

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Fig. 8. Schematic representation of MDCK cell damage produced by A. mauritaniensis T4D (1) Cytopathic effect of amoebic proteases on epithelial cells, (2) disruption of the TJ proteins and increase in cellular permeability, and (3) protection of epithelial cells by the serine protease inhibitor PMSF.

proteins ZO-1, claudin-1 and occludin in MDCK cells, similar to the results reported for A. royreba, A. castellanii, N. fowleri and Entamoeba histolytica, in which the loss of the continuous pattern of TJ proteins in epithelial and human brain endothelial cells (HBMEC) was observed [14,27,36,44]. We also corroborated the degradation of TJ proteins by Western blotting; we observed that ZO-1, claudin-1 and occludin displayed a gradual degradation, as reported for A. castellanii and N. fowleri in MDCK cells and HBMEC [14,27,36]. Moreover, we evaluated whether the degradation of TJ proteins is due to serine proteases; we used PMSF to abolish the cytopathic effect and observed an improvement in the TEER and a reduction in TJ protein degradation. Our results showed that serine proteases participate in the pathogenic behavior of A. mauritaniensis T4D; similar results were reported for A. castellanii, where the use of protease inhibitors prevented damage to the monolayer [25]. From these results, we can conclude that A. mauritaniensis T4D can release proteolytic enzymes that participate in damaging MDCK cells. Moreover, it is necessary to perform further studies to evaluate the cytopathic effect and the alterations in TJ proteins induced by CM in MDCK cells and to evaluate the possible inhibition of the secreted proteases using different protease inhibitors, such as PMSF. In summary, our results demonstrate that the environmental isolate A. mauritaniensis T4D, which was previously characterized as a nonpathogenic amoeba [3], is able to produce and secrete serine proteases that can be involved in epithelial damage and in the alteration of TJ proteins, which could lead to host invasion by amoebae (Fig. 8); further studies are needed to elucidate whether this environmental isolate of A. mauritaniensis T4D is able to induce in vivo damage to the hamster cornea [12].

strain A. castellanii and could induce substantial damage in human and hamster corneal tissues, as it has been reported for A. castellanii and A. polyphaga [12,13]. Based on these results, we evaluated both the cytopathic effect induced by the amoebae and the integrity of TJ proteins in MDCK monolayers, which is a widely reported epithelial model used to analyze the damage induced by FLA, such as A. castellanii, A. culbertsoni, A. royreba [25,36], and Naegleria fowleri [27]. Moreover, this epithelial model allows us to mimic the skin and corneal infections caused by pathogenic Acanthamoeba presented in human cases. Our results showed that A. mauritaniensis T4D is able to induce substantial cytopathic damage to MDCK cells, similar to that reported for A. castellanii, A. culbertsoni and A. royreba strains [37,38]. It is known that A. castellanii has serine proteases, which are considered a virulence factor [10,39]. Therefore, we questioned whether A. mauritaniensis T4D possessed similar protease activities, and we performed a partial biochemical analysis of the proteases of A. mauritanienesis T4D. The type of proteases present in this amoeba was determined using specific protease inhibitors. Our results showed that PMSF was the main protease inhibitor that abolished serine protease activity using Azocoll at pH values of 5, 7 and 9 at 37 °C, as was reported for other Acanthamoeba species [25,39–41]. Other inhibitors of cysteine and metalloproteases can also inhibit the activity of other proteases, although to a lesser degree, as reported by Serrano-Luna et al, (2006) [25] in both TCE and CM of A. castellanii and A. polyphaga. These results suggest that A. mauritaniensis T4D isolated from soil is able to activate proteolytic enzymes at 37 °C, which could participate in host damage. Moreover, we observed by gelatin zymography under physiological conditions (pH 7 and 37 °C) similar to the pH range described for skin (pH values of 4–6) and for human eyes (pH values of 7.3–7.8) [42,43] that TCE and CM from A. mauritaniensis T4D produced remarkable proteolytic activities similar to those reported by SerranoLuna et al, (2006) [25] for the pathogenic strains A. castellanii and A. polyphaga. Interestingly, some proteolytic bands of A. mauritaniensis T4D may correlate with collagenases previously reported from keratitis cases by De Souza-Carvalho et al, 2011 [39]. Finally, we evaluated whether A. mauritaniensis T4D was capable of modifying TJ proteins and decreasing the TEER in MDCK cells via protease activity. We observed that amoebae were able to induce the rearrangement of the TJ

Declaration of Competing Interest The authors declare that they have no competing interests. Acknowledgments The authors acknowledge Dr. Fernando Lares-Villa from Instituto Tecnológico de Sonora for his help in genotyping Acanthamoeba mauritaniensis and MSc Jaime Escobar-Herrera for his technical 10

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assistance in confocal microscopy. This work was supported by the CONACyT grant number 237523.

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References [25] [1] G.S. Visvesvara, H. Moura, F.L. Schuster, Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea, FEMS Immunol. Med. Microbiol. 50 (1) (2007) 1–26, https://doi. org/10.1111/j.1574-695X.2007.00232.x. [2] R. Siddiqui, N.A. Khan, Biology and pathogenesis of Acanthamoeba, Parasit. Vectors 5 (6) (2012) 1–13, https://doi.org/10.1186/1756-3305-5-6. [3] J.F. De Jonckheere, Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp, Appl. Environ. Microbiol. 39 (4) (1980) 681–685. [4] Z. Cao, D.M. Jefferson, N. Panjwani, Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba, J. Biol. Chem. 273 (25) (1998) 15838–15845, https://doi.org/10.1074/jbc.273.25.15838. [5] D.A. Pettit, J. Williamson, G.A. Cabral, F. Marciano-Cabral, In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study, J. Parasitol. 82 (5) (1996) 769–777, https://doi.org/10.2307/ 3283890. [6] M. Michalek, F.D. Sonnichsen, R. Wechselberger, A.J. Dingley, C.W. Hung, A. Kopp, H. Wienk, M. Simanski, R. Herbst, I. Lorenzen, F. Marciano-Cabral, C. Gelhaus, T. Gutsmann, A. Tholey, J. Grotzinger, M. Leippe, Structure and function of a unique pore-forming protein from a pathogenic Acanthamoeba, Nat. Chem. Biol. 9 (1) (2013) 37–42, https://doi.org/10.1038/nchembio.1116. [7] M.M. Mitra, H. Alizadeh, R.D. Gerard, J.Y. Niederkorn, Characterization of a plasminogen activator produced by Acanthamoeba castellanii, Mol. Biochem. Parasitol. 73 (1–2) (1995) 157–164, https://doi.org/10.1016/0166-6851(94) 00109-Z. [8] E. Hadas, T. Mazur, Proteolytic enzymes of pathogenic and non-pathogenic strains of Acanthamoeba spp, Trop. Med. Parasitol. 44 (3) (1993) 197–200. [9] K. Mitro, A. Bhagavathiammai, O.M. Zhou, G. Bobbett, J.H. McKerrow, R. Chokshi, B. Chokshi, E.R. James, Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga, Exp. Parasitol. 78 (4) (1994) 377–385, https://doi.org/ 10.1006/expr.1994.1041. [10] N.A. Khan, E.L. Jarroll, N. Panjwani, Z. Cao, T.A. Paget, Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba, J. Clin. Microbiol. 38 (8) (2000) 2858–2861. [11] A. Ferrante, E.J. Bates, Elastase in the pathogenic free-living amoebae Naegleria and Acanthamoeba spp, Infect. Immun. 56 (12) (1988) 3320–3321. [12] M. Omaña-Molina, F. Navarro-Garcia, A. González-Robles, J. Serrano-Luna Jde, R. Campos-Rodríguez, A. Martínez-Palomo, V. Tsutsumi, M. Shibayama, Induction of morphological and electrophysiological changes in hamster cornea after in vitro interaction with trophozoites of Acanthamoeba spp, Infect. Immun. 72 (6) (2004) 3245–3251, https://doi.org/10.1128/IAI.72.6.3245-3251.2004. [13] M. Omaña-Molina, A. González-Robles, L.I. Salazar-Villatoro, A.R. Cristóbal-Ramos, M. González-Lázaro, E. Salinas-Moreno, R. Méndez-Cruz, M. Sánchez-Cornejo, E. De la Torre-González, A. Martínez-Palomo, Acanthamoeba castellanii: morphological analysis of the interaction with human cornea, Exp. Parasitol. 126 (1) (2010) 73–78, https://doi.org/10.1016/j.exppara.2010.02.004. [14] N.A. Khan, R. Siddiqui, Acanthamoeba affects the integrity of human brain microvascular endothelial cells and degrades the tight junction proteins, Int. J. Parasitol. 39 (14) (2009) 1611–1616, https://doi.org/10.1016/j.ijpara.2009.06.004. [15] H.M. Elsheikha, N.A. Khan, Protozoa traversal of the blood-brain barrier to invade the central nervous system, FEMS Microbiol. Rev. 34 (4) (2010) 532–553, https:// doi.org/10.1111/j.1574-6976.2010.00215.x. [16] L. Shen, Tight junctions on the move: molecular mechanisms for epithelial barrier regulation, Ann. N. Y. Acad. Sci. 1258 (2012) 9–18, https://doi.org/10.1111/j. 1749-6632.2012.06613.x. [17] L. González-Mariscal, A. Betanzos, P. Nava, B.E. Jaramillo, Tight junction proteins, Prog. Biophys. Mol. Biol. 81 (1) (2003) 1–44, https://doi.org/10.1016/S00796107(02)00037-8. [18] J.R. Turner, Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application, Am. J. Pathol. 169 (6) (2006) 1901–1909, https://doi.org/ 10.2353/ajpath.2006.060681. [19] F. Rivera, F. Medina, P. Ramírez, J. Alcocer, G. Vilaclara, E. Robles, Pathogenic and free-living protozoa cultured from the nasopharyngeal and oral regions of dental patients, Environ. Res. 33 (2) (1984) 428–440, https://doi.org/10.1016/00139351(84)90040-9. [20] F. Castro-Muñozledo, Development of a spontaneous permanent cell-line of rabbit corneal epithelial-cells that undergoes sequential stages of differentiation in cellculture, J. Cell Sci. 107 (1994) 2343–2351. [21] M.T. Ortíz-Melo, E. Sánchez-Guzman, A. González-Robles, J. Valdes, E. GomezFlores, F. Castro-Muñozledo, Expression of claudins-2 and-4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier, Biol. Open 2 (2) (2013) 132–143, https://doi.org/10.1242/bio.20123145. [22] J.M. Schroeder, G.C. Booton, J. Hay, I.A. Niszl, D.V. Seal, M.B. Markus, P.A. Fuerst, T.J. Byers, Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge, J. Clin. Microbiol. 39 (5) (2001) 1903–1911, https://doi.org/10. 1128/JCM.39.5.1903-1911.2001. [23] P.A. Fuerst, G.C. Booton, M. Crary, Phylogenetic analysis and the evolution of the

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

11

18S rRNA gene typing system of Acanthamoeba, J. Eukaryot. Microbiol. 62 (1) (2015) 69–84, https://doi.org/10.1111/jeu.12186. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254, https://doi.org/10.1016/0003-2697(76)90527-3. J. Serrano-Luna, I. Cervantes-Sandoval, J. Calderón, F. Navarro-García, V. Tsutsumi, M. Shibayama, Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii, Can. J. Microbiol. 52 (1) (2006) 16–23, https://doi.org/ 10.1139/w05-114. R.A. McCloy, S. Rogers, C.E. Caldon, T. Lorca, A. Castro, A. Burgess, Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events, Cell Cycle 13 (9) (2014) 1400–1412, https://doi.org/10.4161/cc.28401. M. Shibayama, M. Martínez-Castillo, A. Silva-Olivares, S. Galindo-Gómez, F. Navarro-García, J. Escobar-Herrera, M. Sabanero, V. Tsutsumi, J. Serrano-Luna, Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri, Microbiol 159 (Pt 2) (2013) 392–401, https://doi.org/10.1099/mic.0.063255-0. Y.G. He, J.Y. Niederkorn, J.P. McCulley, G.L. Stewart, D.R. Meyer, R. Silvany, J. Dougherty, In vivo and in vitro collagenolytic activity of Acanthamoeba castellanii, Invest. Ophthalmol. Vis. Sci. 31 (11) (1990) 2235–2240. H. Trabelsi, A. Sellami, F. Dendena, H. Sellami, F. Cheikh-Rouhou, F. Makni, D.S. Ben, A. Ayadi, Free-living amoebae (FLA): morphological and molecular identification of Acanthamoeba in dental unit water, Parasite 17 (1) (2010) 67–70, https://doi.org/10.1051/parasite/2010171067. F.L. Schuster, G.S. Visvesvara, Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals, Int. J. Parasitol. 34 (9) (2004) 1001–1027, https://doi.org/10.1016/j.ijpara.2004.06.004. D.I. Chung, H.S. Yu, M.Y. Hwang, T.H. Kim, T.O. Kim, H.C. Yun, H.H. Kong, Subgenus classification of Acanthamoeba by riboprinting, Korean J. Parasitol. 36 (2) (1998) 69–80 https://doi.org/10.3347/kjp.1998.36.2.69. J.F. De Jonckheere, Isoenzyme and Total protein-analysis by agarose isoelectricfocusing, and taxonomy of the genus Acanthamoeba, J. Protozool. 30 (4) (1983) 701–706, https://doi.org/10.1111/j.1550-7408.1983.tb05346.x. A.K. Tice, L.L. Shadwick, A.M. Fiore-Donno, S. Geisen, S. Kang, G.A. Schuler, F.W. Spiegel, K.A. Wilkinson, M. Bonkowski, K. Dumack, D.J. Lahr, E. Voelcker, S. Clauss, J. Zhang, M.W. Brown, Expansion of the molecular and morphological diversity of Acanthamoebidae (Centramoebida, Amoebozoa) and identification of a novel life cycle type within the group, Biol. Direct 11 (1) (2016) 69, https://doi. org/10.1186/s13062-016-0171-0. N.A. Khan, Acanthamoeba: biology and increasing importance in human health, FEMS Microbiol. Rev. 30 (4) (2006) 564–595, https://doi.org/10.1111/j.15746976.2006.00023.x. M. Omaña-Molina, D. Hernandez-Martinez, R. Sanchez-Rocha, U. Cardenas-Lemus, C. Salinas-Lara, A.R. Mendez-Cruz, L. Colin-Barenque, P. Aley-Medina, J. EspinosaVillanueva, L. Moreno-Fierros, J. Lorenzo-Morales, In vivo CNS infection model of Acanthamoeba genotype T4: the early stages of infection lack presence of host inflammatory response and are a slow and contact-dependent process, Parasitol. Res. 116 (2) (2017) 725–733 https://10.1007/s00436-016-5338-1. C. Flores-Maldonado, A. González-Robles, L. Salazar-Villatoro, M. Omaña-Molina, J.M. Gallardo, M. González-Lazaro, V.I. Hernández-Ramirez, P. Talamás-Rohana, J. Lorenzo-Morales, A. Martínez-Palomo, 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 (2017) 69–75, https://doi.org/10.1016/j.exppara.2017.10.013. A. González-Robles, G. Castanon, A.R. Cristobal-Ramos, A. Lazaro-Haller, M. Omaña-Molina, P. Bonilla, A. Martínez-Palomo, Acanthamoeba castellanii: structural basis of the cytopathic mechanisms, Exp. Parasitol. 114 (3) (2006) 133–140, https://doi.org/10.1016/j.exppara.2006.02.023. M. Omaña-Molina, A. González-Robles, L. Iliana Salazar-Villatoro, J. LorenzoMorales, A.R. Cristobal-Ramos, V.I. Hernández-Ramirez, P. Talamás-Rohana, A.R. Mendez Cruz, A. Martínez-Palomo, Reevaluating the role of Acanthamoeba proteases in tissue invasion: observation of cytopathogenic mechanisms on MDCK cell monolayers and hamster corneal cells, Biomed. Res. Int. 2013 (2013) 461329, , https://doi.org/10.1155/2013/461329. F.R. de Souza Carvalho, L.C. Carrijo-Carvalho, A.M. Chudzinski-Tavassi, A.S. Foronda, D. de Freitas, Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis, Clin. Microbiol. Infect. 17 (4) (2011) 603–609, https://doi.org/10.1111/j.1469-0691. 2010.03252.x. V.P. Sant'ana, L.C. Carrijo-Carvalho, A.S. Foronda, A.M. Chudzinski-Tavassi, D. de Freitas, F.R. de Carvalho, Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp, Graefes Arch. Clin. Exp. Ophthalmol. 253 (1) (2015) 65–75, https://doi.org/10.1007/s00417-014-2783-3. J.L. Harrison, G.A. Ferreira, E.S. Raborn, A.D. Lafrenaye, F. Marciano-Cabral, G.A. Cabral, Acanthamoeba culbertsoni elicits soluble factors that exert anti-microglial cell activity, Infect. Immun. 78 (9) (2010) 4001–4011, https://doi.org/10. 1128/IAI.00047-10. S.M. Ali, G. Yosipovitch, Skin pH: from basic science to basic skin care, Acta Derm. Venereol. 93 (3) (2013) 261–267, https://doi.org/10.2340/00015555-1531. M.B. Abelson, I.J. Udell, J.H. Weston, Normal human tear pH by direct measurement, Arch. Ophthalmol. 99 (2) (1981) 301, https://doi.org/10.1001/archopht. 1981.03930010303017. A. Betanzos, R. Javier-Reyna, G. García-Rivera, C. Bañuelos, L. González-Mariscal, M. Schnoor, E. Orozco, The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage, PLoS One 8 (6) (2013) e65100, , https://doi.org/10.1371/journal.pone.0065100.