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Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections [1]

['Helena Lúcia Carneiro Santos', 'Laboratório De Doenças Parasitárias', 'Instituto Oswaldo Cruz Fiocruz', 'Rio De Janeiro', 'Coleção De Protozoários Da Fiocruz', 'Laboratório De Parasitologia', 'Departamento De Patologia Da Faculdade De Medicina Da Universidade Federal Fluminense', 'Gabriela Linhares Pereira', 'Rhagner Bonono Do Reis', 'Igor Cardoso Rodrigues']

Date: 2025-01 

Abstract Leishmaniasis represents a severe global health problem. In the last decades, there have been significant challenges in controlling this disease due to the unavailability of licensed vaccines, the high toxicity of the available drugs, and an unrestrained surge of drug-resistant parasites, and human immunodeficiency virus (HIV)–Leishmania co-infections. Leishmania spp. preferentially invade macrophage lineage cells of vertebrates for replication after subverting cellular functions of humans and other mammals. These early events in host–parasite interactions are likely to influence the future course of the disease. Thus, there is a continuing need to discover a simple cellular model that reproduces the in vivo pathogenesis. Acanthamoeba spp. are non-mammalian phagocytic amoeba with remarkable similarity to the cellular and functional aspects of macrophages. We aimed to assess whether the similarity reported between macrophages and Acanthamoeba spp. is sufficient to reproduce the infectivity of Leishmania spp. Herein, we analyzed co-cultures of Acanthamoeba castellanii or Acanthamoeba polyphaga with Leishmania infantum, Leishmania amazonensis, Leishmania major, and Leishmania braziliensis. Light and fluorescence microscopy revealed that the flagellated promastigotes attach to the A. castellanii and/or A. polyphaga in a bipolar and or random manner, which initiates their uptake via pseudopods. Once inside the cells, the promastigotes undergo significant changes, which result in the obligatory amastigote-like intracellular form. There was a productive infection with a continuous increase in intracellular parasites. However, we frequently observed intracellular amastigotes in vacuoles, phagolysosomes, and the cytosol of Acanthamoeba spp. Our findings corroborate that Leishmania spp. infects Acanthamoeba spp. and replicates in them but does not cause their rapid degeneration or lysis. Overall, the evidence presented here confirms that Acanthamoeba spp. have all prerequisites and can help elucidate how Leishmania spp. infect mammalian cells. Future work exposing the mechanisms of these interactions should yield novel insights into how these pathogens exploit amoebae.

Author summary Leishmaniasis represents a severe global health problem, and drug resistance is a growing concern. Leishmania spp. are obligate intracellular parasites that survive within cells of the vertebrate macrophage, modulating their activation. Understanding the multilayered relationship between metabolism and function of innate immune cells during infection has great therapeutic and preventive potential. Mammalian macrophages and Acanthamoeba spp. display similarities in the molecular mechanisms involved in directional motility, recognition, binding, engulfment, and the phagolysosomal processes, and they express similar receptors. Hence, we hypothesize that Acanthamoeba spp. represent a model that can be used to evaluate macrophage–pathogen interactions from the perspective of innate immunity. However, it has not yet been described whether Leishmania spp. can survive in Acanthamoeba spp. We found that Acanthamoeba spp. support Leishmania growth. Acanthamoeba spp. contain ancient pathogen recognition mechanisms, and Leishmania spp. could manipulate amoeboid functions to favor their survival and replication based on strategies of ancestral metabolic pathways. Our robust evidence highlights that amoebae could be used as a model to understand the biology and evolution of host–Leishmania interactions.

Citation: Santos HLC, Pereira GL, do Reis RB, Rodrigues IC, Masini d’Avila C, Vidal VE (2024) Using Acanthamoeba spp. as a cell model to evaluate Leishmania infections. PLoS Negl Trop Dis 18(10): e0012517. https://doi.org/10.1371/journal.pntd.0012517 Editor: Naveed Ahmed Khan, University of Derby, UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND Received: September 10, 2023; Accepted: September 6, 2024; Published: October 2, 2024 Copyright: © 2024 Santos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Instituto Oswaldo Cruz intramural funding (PAEF II-IOC-23-FIO-18-2-53), and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) Project ID: N° DO PROCESSO SEI-260003/002148/2021 - APQ1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Leishmaniasis is a term used to define a wide-ranging group of diseases caused by protozoa of the genus Leishmania [1,2]. These infections result in a broad spectrum of clinical manifestations that range from asymptomatic infection to localized, disseminated ulcers, sometimes with mucosal involvement (cutaneous leishmaniasis), or affect internal organs, such as the liver, spleen, and bone marrow (visceral leishmaniasis), which can be fatal if left untreated [3,4]. In recent years, leishmaniasis has been considered a neglected and emerging group of diseases. These diseases constitute a global grave public-health problem that leads to great suffering and high socio-cultural and economic costs [5]. There is no available vaccine and drug resistance is emerging [6]. Additionally, the available drugs have severe side effects, further complicating treatment, and cannot eliminate the pathogen [6]: The parasite remains in the body and can cause a relapse when there is immunosuppression [6,7]. According to Word Health Organization (WHO), over 1 billion people are at risk of infection in high-burden countries [8,9]. There are an estimated 700,000 to 1 million new cases each year, highlighting the importance of this public health problem [10]. In a vertebrate host, Leishmania parasites can evade the defense barriers of the host and even benefit from them [9]. Indubitably, macrophages are key cells to the body’s guards against pathogens [10]. However, Leishmania spp. have evolved to modulate vacuole biogenesis to generate a unique intravacuolar niche that is suitable for their proliferation inside macrophages [4,11]. In this niche, Leishmania differentiates into the amastigote form that inhabits digestive vacuoles, which fuse to macrophage lysosomes, generating phagolysosomes [12]. Elucidating the complex Leishmania–macrophage interactions is crucial to prevent and treat leishmaniasis [11,12]. To survive successfully and multiply within macrophages, Leishmania must undergo profound biochemical and morphological adaptations to enter a host cell, establish an infection, and evade or modulate the subsequent immune response [13,14]. These abilities might have resulted from cellular and molecular adaptations to the ancestral phagocytosis process [15]. Mammalian macrophages and Acanthamoeba spp. display similarities in the molecular mechanisms involved in directional motility, recognition, binding, engulfment, and phagolysosome processing of bacteria and fungi [16]. They also display conserved pathogen-recognition mechanisms [16,17]. Acanthamoeba spp. are opportunistic protozoans, ubiquitously distributed in nature, that are mainly found in soil and water. In general, the life cycle of these amoeba consists of two stages, trophozoite and cyst. Trophozoites are the motile forms that mainly feeds on bacteria, algae, yeast, or small organic particles via phagocytosis or pinocytosis [16,18]. Trophozoites closely resemble the internal structure of a mammalian cell. They contain various organelles such as mitochondria, ribosomes, centrosomes, vacuoles, and the Golgi apparatus. Vacuoles are a critical component of trophozoites: they include the contractile vacuole, which expels water for osmotic regulation, lysosomes, digestive vacuoles, and many glycogen-containing vacuoles [18].Over the past several decades, Acanthamoeba spp. have gained increasing attention due to their ability to act as a host/reservoir for microbial pathogens, particularly their role in causing damage and sometimes fatal human and animal infections [16]. Recent studies suggest that Acanthamoeba–microbe adaptations are considered an evolutionary model of macrophage–pathogen interactions and acts as a “genetic melting pot” with cross-species exchange of genes [17]. Due to their remarkable cellular and functional similarities, authors have speculated that Acanthamoeba spp. and macrophages are evolutionarily related [15,18]. An exciting hypothesis is Acanthamoeba spp. may be an in vitro model to investigate pathogen–macrophage interactions [19]. Phagocytosis is a complex process that has been maintained during evolution [15,20]. Acanthamoeba spp. are well known for their exuberant motility and active pseudopodia when interact with bacteria, viruses, protists, and fungi [17,19]. Acanthamoeba spp. can harbor important human pathogens such as Legionella pneumophila, Aeromonas spp., Pseudomonas spp., Escherichia coli, and Mycobacterium spp., and others cultured from clinical specimens [19,21–23]. It is noteworthy that some of these pathogens can subvert the microbicidal activity and replicate inside Acanthamoeba spp. with mechanisms that are similar to those used for survival within macrophages [24–26]. While Acanthamoeba spp. and macrophages take up microbes via phagocytosis, whether these distinct hosts have an evolutionary or convergent evolution remains unknown. Moreover, not all the phagocytic pathways are totally conserved between amoebae and macrophages, the endocytic system evasion mechanisms such as inhibition of phagosome–lysosome fusion, are common to escape from either host [19]. Despite efforts to unravel the mechanisms of Leishmania pathogenicity and the risk of infection, and to develop novel treatments and vaccines against the parasite, there are still gaps in the state-of-the-art that need be explored. The innate immunity pathways are widely conserved in nature, and evaluation of Acanthamoeba–Leishmania interactions could represent an important model to understand the mechanisms of virulence and host immunity. We evaluated whether Acanthamoeba spp. are similar enough to macrophages to reproduce Leishmania spp. infectivity. Understanding these interactions will potentially reveal new mechanisms for investigating host–parasite interactions.

Material and Methods Schematic overview of Leishmania-Acanthamoeba interaction assay Acanthamoeba stains and cultivation conditions. Two Acanthamoeba spp. strains were used in this study: an environmental strain Acanthamoeba castellanii designated as Neff (ATCC 30010), and Acanthamoeba polyphaga (ATCC 30431) that was isolated from corneal scrapings of a patient with acute ulceration of the right eye. The amoebae were grown in Peptone Yeast Glucose (PYG) medium containing 0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract, and 1.5% (w/v) glucose as a monolayer in 25 cm2 tissue culture flasks at 27°C. When cells formed a monolayer, the trophozoites were harvested, centrifuged (200 g for 5 min), washed three times with phosphate-buffered saline (PBS), and suspended in PYG broth or PBS. Cells were counted in a hemocytometer chamber, and the viability was assessed by the trypan blue exclusion assay. Prior to the experiments, the amoebae were grown in tissue culture flasks at 27°C without shaking, and the media refreshed after 17–20 h. In addition, the influence of culture media (Grace’s insect cell culture medium (Grace’s), liver infusion tryptose [LIT], and Schneider’s insect medium [Schneider’s], without supplementation with fetal calf serum [FCS]) on the growth and viability of Acanthamoeba spp. was assessed. Acanthamoeba spp growth was monitored daily for 6 days with a Neubauer chamber. Viability was assessed by mobility and lack of staining after challenging with trypan blue. All results correspond to three independent assays that were run in duplicates. Before starting the experiments, parasites were maintained in each medium for a minimum of three passages. Leishmania strains and cultivation conditions of promastigotes. Three causative species of cutaneous leishmaniasis, Leishmania amazonensis (MHOM/BR/PH8), Leishmania braziliensis (strain Thor, MCAN/BR/1998/619), and Leishmania major (MHOM/SU/1973/5-ASKH), and one causative species of visceral leishmaniasis, Leishmania infantum (MHOM/BR/1974/PP75), were used in this study. Promastigotes were obtained from the Protozoa Culture Collection (COLPROT) and the Leishmania Collection (CLIOC) from Instituto Oswaldo Cruz–Fundação Oswaldo Cruz (IOC-FIOCRUZ) and cultured in Schneider’s medium (pH 7.2) supplemented with 20% fetal bovine serum (FBS), streptomycin (40 μg/mL), and penicillin (100 U/mL) at 27°C. The parasites were maintained by serial passaging twice per week. Before starting the experiments, promastigotes were sub-passaged to a fresh medium every 4 days to maintain the growth and viability of the parasites. Co-culture of Leishmania spp. and Acanthamoeba spp. For light microscopy analyses, A. castellanii and A. polyphaga were seeded in eight-well chamber Lab-Tek chambers (Nunc, Roskilde, Denmark) at 1 × 105 cells per well. Then, 1 × 106 promastigotes (L. braziliensis, L. amazonensis, L. infantum, and L. major) in the stationary growth phase of the respective culture media (1:10 ratio) were added to two wells each at 37°C or 27°C. After 3, 6, 12, 24, 36, 72 h, and 96 h, the supernatant from each chamber slide well was discarded and the top of the plate was removed. Non-adhered promastigotes were removed by three washing cycles with PBS (pH 7.2). The attached cells were fixed with methanol before being submitted to panoptic fast staining. To evaluate infectivity, the slides were analyzed under an optical microscope (Olympus Optical) at 1000× total magnification using immersion oil. For fluorescence microscopy analyses, promastigotes in the mid-log growth phase were incubated with 0.1% fluorescein isothiocyanate (FITC) in a carbonate-bicarbonate buffer (pH 9.4; 9.5 mL of 0.2 M Na 2 CO 3 mixed with 41.5 mL of 0.2 M NaHCO 3 ) for 1 h at room temperature in the dark, based on a previously described method [27]. Finally, the pellet was resuspended in 1 mL of PBS. A. castellanii and A. polyphaga were seeded in eight-well chamber Lab-Tek chambers (Nunc, Roskilde, Denmark) at a density of 1 × 105 cells per well. Then, 1 × 106 FITC-labeled promastigotes forms (L. braziliensis, L. amazonensis, L. infantum, and L. major) in the stationary growth phase of the respective culture media (1:10 ratio) were added to two wells each, as described above. Each dried slide was fixed with absolute methanol and examined under a fluorescence microscope (Zeiss Axiolab) at 400× and 1000× total magnification using immersion oil (Fig 1). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Schematic overview of Leishmania-Acanthamoeba interaction assay. The host cells (Acanthamoeba castellanii or Acanthamoeba polyphaga) were plated, then infected by Leishmania spp promastigotes transformed in amastigotes of each species with a multiplicity of infection (MOI) of 1:10. Three, six, 12, 24, 36, 72 h, and 96 h post-infection (HPI), cells were fixed and stained with Panoptic fast staining and visually analyzed. These observations were further confirmed by fluorescence microscopic imaging of macrophages infected with FITC-labeled Leishmania spp. https://doi.org/10.1371/journal.pntd.0012517.g001 The parasite release and differentiation assay. Twenty-four hours after infection, Acanthamoeba monolayers were washed and incubated with 0.01% sodium dodecyl sulfate (SDS; Sigma-Aldrich) for 10 min to allow lysis of Acanthamoeba and the release of amastigotes. Then, cultures were fed with five mL of Schneider’s medium, supplemented with 10% FBS, and incubated at 27°C and for an additional 2 days.

Discussion This report represents the first description of the abilities of L. infantum, L. amazonensis, L. major, and L. braziliensis to grow and subvert the functions of A. castellanii and A. polyphaga. Like macrophages, intracellular infection of Acanthamoeba spp. by Leishmania promastigotes relies on a series of key events, including attachment, internalization, amastigote differentiation, and intracellular survival [27]. The use of a cell line to study infections in vitro is an essential approach to evaluate the distinct aspects of Leishmania biology as well as its prevention and treatment, which require a clear understanding of the molecular network between parasites and their hosts [28]. Understanding the interactions would allow us to combat the disease by developing new drugs and therapies or by identifying important parasite antigens that could be exploited to develop vaccines. There have been several reports of Leishmania parasitizing cells other than macrophages [29, 30]; however, it is recognized that in a vertebrate host, Leishmania spp. mainly resides within cells of the macrophage lineage [30]. Macrophages from different origins are amenable to successful Leishmania infection in vitro [31, 32]. These cells have been a mainstay of leishmaniasis research for more than 40 years, although there are many knowledge gaps. The main challenge is to elucidate that mechanisms lead to the subversion of macrophages [33]. Designing and applying a physiologically robust in vitro assay is highly relevant for drug screening and evaluation of biochemistry, physiology, and metabolism. Macrophages represent the major effector cells that phagocytose parasites within vertebrate hosts [30]. The interaction between the innate and adaptive immune response is a crucial factor in the process of differentiating macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, leading to resistance or susceptibility to Leishmania infection. Besides, various immune cells, including T cells, play a significant role in modulating macrophage polarization by releasing cytokines that influence macrophage maturation and function. Other immune cells can also influence macrophage polarization in a T cell–independent manner [32,33]. The host’s immune responses and individual variations in parasite strains may be critical to determine the clinical outcome of leishmaniasis in a patient [31]. The influence of the innate immune response during Leishmania infection has been proposed to favor parasite survival, allowing it to fully establish itself within the mammalian host [30,34]. In the present study, Acanthamoeba spp. were similar to macrophages in that they were permissible to Leishmania spp., but they would have no influence on the adaptive immune response. Interestingly, Acanthamoeba spp. are well-characterized non-mammalian phagocytic cells with remarkable similarity to macrophages. Indeed, the similar traits and features of macrophages and Acanthamoeba spp.—including the ancient process of phagocytosis—provide evidence of a common ancestry [15,35] or convergent evolution. Mammalian macrophages and Acanthamoeba spp. display similarities in the molecular mechanisms involved in directional motility, recognition, binding, engulfment, and phagolysosome processes, and express common primitive receptors [16,17,36] Like macrophages, some microorganisms have evolved strategies for efficient uptake by Acanthamoeba, and they are able to regulate phagosome maturation to make the phagosome more hospitable for growth and to avoid destruction [19]. Acanthamoeba spp. are considered hosts for bacteria, viruses, yeast, and protozoa [37,38]. Their genome includes several genes that encode pattern recognition receptors (e.g., lipopolysaccharide-binding protein/bactericidal permeability enhancing protein [LBP/BPI], C-lectin, C-type lectin, mannose-binding protein [MBP], D-galactoside/L-rhamnose-containing lectin SUEL domain, NB-ARC tetratricopeptide containing repeat-containing protein ERVR, and endogenous virus receptor) [39]. These receptors allow amoeba to interact with a vast array of species. Over the past several decades, Acanthamoeba spp. have received increasing attention due to their ability to act as a host/reservoir for microbes, including viruses (mimivirus, coxsackieviruses, adenoviruses, poliovirus, echovirus, enterovirus, vesicular stomatitis virus, etc.), bacteria (Aeromonas, Bacillus, Bartonella, Burkholderia, Campylobacter, Chlamydophila, Coxiella, E. coli, Flavobacterium, Helicobacter, Legionella, Listeria, Staphylococcus, Mycobacterium, Pasteurella, Prevotella, Porphyromonas, Pseudomonas, Rickettsia, Salmonella, Shigella, Vibrio, etc.), protists (Cryptosporidium and Toxoplasma gondii), and yeast (Cryptococcus, Blastomyces, Sporothrix, Histoplasma, Streptomyces, Exophiala, etc.) [16,17,40]. Moreover, several pathogens can survive within A. castellanii by using similar strategies as when they encounter macrophages [16,23,41–44]. A survey showed that the Dot/Icm type IV secretion system is required by L. pneumophila for intracellular proliferation within human macrophages and Acanthamoeba [43]. Moreover, L. pneumophila is taken up by Acanthamoeba by coiling phagocytosis but can impede phagosome–lysosome fusion. In turn, Vibrio cholerae can resist amoeboid phagosome killing by neutralizing proteolytic enzymes, changing the local pH, and counteracting reactive nitrogen and oxygen species that may otherwise kill internalized bacteria [45]. Likewise, invasion of A. castellanii or macrophages by Mycobacterium spp. shows notable similarities at the transcriptional and post-translational levels [46]. A. castellanii can also engulf fungi that are potential pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitides, Fusarium solani, and Sporothrix schenckii [47]. Mannose-binding lectins (MBLs) play a critical role in activating antifungal responses by immune cells [48,49]. A study has suggested that mannose-binding proteins identified in fungal recognition by both amoeboid and macrophage surfaces belong to the Conavalin A-like superfamily. Yet, the results reveal a limitation: a short sequence of 53 amino acids does not have enough resolution to predict protein folding accurately. [36]. However, data from the literature show us that macrophages and Acanthamoeba may share similar interaction mechanisms with fungal and bacteria pathogens, indicating a possible convergent evolution. We found that L. braziliensis, L. Amazonensis, L. infantum and L. major could infect and grow within A. castellanii and A. polyphaga. These amoebae engulfed promastigotes, permitted the intracellular conversion of the promastigotes to an amastigote-like state, and supported their intracellular multiplication. There was a visible increase in the number of parasites per infected Acanthamoeba and in the total number of parasites per monolayer (although the number of parasites were not quantified). We evaluated attachment and infection primarily by inspecting fixed specimens from co-cultures of promastigotes and Acanthamoeba spp. A potential pitfall in this method is the possibility that some of the attached parasites might have been superimposed on the Acanthamoeba image and thus erroneously scored as phagocytosed. Despite this limitation, our findings provide novel insight into host–leishmania interactions. A. castellanii showed slow growth over 6 days in LIT and Schneider’s medium but remained viable and the death phase was not detected. Schneider’s insect medium (Schneider’s) and Grace’s insect cell culture medium (Grace’s) are defined media and are indicated for the continuous in vitro cultivation of promastigote forms of Leishmania [50]. However, a study demonstrated that the supplementation of LIT culture medium with fetal calf serum is the most suitable strategy to cultivate L. infantum parasites enabling the maintenance of growth and infective parasites for research uses. The authors showed that promastigote forms have a better growth in LIT and Schneider’s with or without FCS when compared to that in Grace’s [51]. In nature, promastigotes may be inoculated at a cutaneous site where complement is not active. Acanthamoeba spp. are lower eukaryotes that utilize a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms [39]. Many studies have shown that the interaction between the innate and adaptive immune responses is a crucial factor leading to resistance or susceptibility to Leishmania infection [52]. It is conceivable that interfering with these processes represents a promising new strategy for antileishmanial intervention. In the present study, Leishmania spp. could subvert the functions of A. castellanii and A. polyphaga based on the innate immune response. Leishmania spp. could manipulate amoeboid functions to favor their survival and replication based on strategies of ancestral metabolic pathways. These novel findings indicate that Leishmania spp. and Acanthamoeba spp. co-cultures are a relatively inexpensive and easy-to-control model system for which multiple variables can be controlled. This cell model might provide new insights into how Leishmania infects cells and new therapeutic avenues to manipulate the innate immune response to protect cells from Leishmania infection.

Conclusion We have shown that L. braziliensis, L. amazonensis, L. infantum, and L. major are amoeba-resistant flagellates and that A. castellanii and A. polyphaga can be useful as a model to evaluate interactions for Leishmania species. Our findings contribute to the knowledge about the molecular aspects of the Leishmania–Acanthamoeba interaction considering innate immunity. The identification of promising molecules involved during phagocytosis and subversion of Leishmania spp. in amoeba could be extrapolated to macrophages. Thus, this cellular model should contribute to the understanding of the yet-to-be-elucidated mechanisms that are important to combat leishmaniasis.

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