Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture

Garajová, Mária; Mrva, Martin; Vaškovicová, Naděžda; Martinka, Magdalena; Melicherová, Janka; Valigurová, Andrea Bardůnek

doi:10.1038/s41598-019-41084-6

Cited by 42 publications

(36 citation statements)

References 73 publications

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“…Acanthamoeba cysts have an ectocyst wall (the outer cyst wall) consisting of acid-insoluble protein-containing materials and an endocyst wall (the inner cyst wall), which is partially composed of cellulose [3,7,8]. Recent studies have shown that cellulose fibrils exist in both cyst walls, and cellulose synthesis has been suggested as an important target for the treatment of Acanthamoeba infections [74]. Previous research on cellulose synthesis inhibitors and the knockdown of Acanthamoeba cellulose synthase has found that cellulose is essential to the formation of cyst walls during encystation [17,75].…”

Section: Discussionmentioning

confidence: 99%

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba

et al. 2020

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Background: The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba. Methods: We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression Results: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed. Conclusions: These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.

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Section: Discussionmentioning

confidence: 99%

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba

et al. 2020

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“…Cyst walls are largely composed of acid-resistant proteins and polysaccharides, among which cellulose is one of the key constituents [13]. Magistrado-Coxen et al [24] demonstrated that purified cyst walls retained an outer ectocyst layer, an inner endocyst layer, and ostioles that connect them.…”

Section: Discussionmentioning

confidence: 99%

“…For cellulose, glycogen phosphorylase is the key enzyme that breaks down glycogen to provide glucose for the synthesis of cellulose [11, 12]. Garajová et al [13] detected clustering of intramembranous particles during encystation. It was suggested that during endocyst formation, intramembranous particle clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that are reduced after cyst wall completion.…”

Section: Introductionmentioning

confidence: 99%

Novel insights into the potential role of ion transport in sensory perception in Acanthamoeba

et al. 2019

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BackgroundAcanthamoeba is well known to produce a blinding keratitis and serious brain infection known as encephalitis. Effective treatment is problematic, and can continue up to a year, and even then, recurrence can ensue. Partly, this is due to the capability of vegetative amoebae to convert into resistant cysts. Cysts can persist in an inactive form for decades while retaining their pathogenicity. It is not clear how Acanthamoeba cysts monitor environmental changes, and determine favourable conditions leading to their emergence as viable trophozoites.MethodsThe role of ion transporters in the encystation and excystation of Acanthamoeba remains unclear. Here, we investigated the role of sodium, potassium and calcium ion transporters as well as proton pump inhibitors on A. castellanii encystation and excystation and their effects on trophozoites.ResultsRemarkably 3′,4′-dichlorobenzamil hydrochloride a sodium–calcium exchange inhibitor, completely abolished excystation of Acanthamoeba. Furthermore, lanthanum oxide and stevioside hydrate, both potassium transport inhibitors, resulted in the partial inhibition of Acanthamoeba excystation. Conversely, none of the ion transport inhibitors affected encystation or had any effects on Acanthamoeba trophozoites viability.ConclusionsThe present study indicates that ion transporters are involved in sensory perception of A. castellanii suggesting their value as potential therapeutic targets to block cellular differentiation that presents a significant challenge in the successful prognosis of Acanthamoeba infections.

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“…In this study, we have demonstrated that in the cyst, the chitin is synthesized by membrane-bound chitin synthase enzyme. Recently, a very similar model for cellulose microfibril synthesis by membrane-bound cellulose synthase has been proposed in Achanthamoeba cyst (36). We believe both the processes are taking place simultaneously in Entamoeba.…”

Section: Localization Of the Chitin Synthase On Cyst Surface Guides Cmentioning

confidence: 99%

Novel insights intoEntamoebacyst wall formation and the importance of actin cytoskeleton

Ghosh

Krishnan

Nayak

2020

Preprint

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21The cyst wall of Entamoeba histolytica, the causative agent of the amoebiasis, is a 22 potential target for new drugs. The "Wattle and Daub" model of cyst wall formation of 23 Entamoeba invadens had already been reported. In this study, we demonstrate in more detail the 24 morphological stages of chitin wall formation in E. invadens using fluorescent chitin-binding 25 dyes and immunolocalization of the cyst wall proteins. Here, the expression and localization of 26 chitin synthase and the importance of actin cytoskeleton dynamics at cellular level, during 27 encystations have been demonstrated for the first time. Chitin deposition was found to be 28 initiated on the cell surface mostly from one distinct point, though multipoint initiation was also 29 observed sometimes. From these points, the wall grew outwards and gradually covered the entire 30 cyst surface with time. The initiation of chitin deposition was guided by the localization of chitin 31 synthase 1 on the plasma membrane. The gradual formation of the cyst wall follows the Wattle 32 and daub model. The chitin deposition occurred on the foundation of Jacob lectin at the cell 33 membrane, and the other cyst wall components, like chitinase, and Jessie were also found to be 34 present in the growing incomplete walls. In contrary to the Wattle and daub model, Jessie was 35 found to be expressed and localized in the growing wall at the early hours of encystations. 36During encystation, F-actin was reorganized into the cortical region within an hour encystation 37 initiation and remained intact until the completion of the chitin wall. Disruption of cortical actin 38 polymerization with 2, 3-Butanedione monoxime inhibited proper wall formation but produced 39 wall-less cysts or cysts with defective chitin wall. Malformations of cyst-walls were mainly due 40 to improper localization and activity of chitin synthases, which indicates the indispensability of 41 cortical actin cytoskeleton for the proper cyst wall formation.42 3 43 Author Summary 44 Entamoeba parasites reach new hosts using the resistant cyst form, so preventing its 45 formation can stop the spread of amoebiasis. The resistant nature of the cyst is due to the chitin 46 wall, and thus identifying the critical steps of wall formation could provide targets for designing 47 new drugs. Here we studied the morphological stages of the cyst wall formation by observing 48 how the chitin and other cell wall components were deposited on the cell surface using 49 fluorescent chitin-binding dyes and antibodies against cyst wall proteins. In most cases, the 50 chitin wall was found to start from one distinct point from which it spread all over the cell 51 surface, guided by chitin synthase. The composition of these incomplete walls was the same as a 52 mature cyst wall indicating that the wall may be a result of extracellular self-assembly of its 53 constituents from one starting point. We have also observed that F-actin polymerized in the 54 cortex of encysting cells and its disruption resulted in wall-less...

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Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture

Cited by 42 publications

References 73 publications

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba

Novel insights into the potential role of ion transport in sensory perception in Acanthamoeba

Novel insights intoEntamoebacyst wall formation and the importance of actin cytoskeleton

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