Novel insights into Entamoeba cyst wall formation and the importance of actin cytoskeleton

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


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Entamoeba histolytica is the causative agent of amoebiasis, the third most important 65 parasitic disease after malaria and schistosomiasis, and causes 40-50 million cases and 40,000-66 100,000 deaths annually (1). Nitroimidazole drugs like metronidazole, tinidazole, ornidazole are 67 the most important drugs for amoebiasis, but most of these drugs have serious side effects. 68 Though drug resistance in clinical isolates has not been reported yet, drug-resistant E. histolytica 69 have been generated in laboratory condition (2,3) indicating the widespread use of anti-amoebic 70 drugs may eventually cause drug resistance. Thus it is necessary to find new, safer drugs, and  Immunization with cyst wall proteins of Giardia lamblia has been reported to cause significant 77 reduction of cyst shedding in murine models (4-6). To find such drug or vaccine candidates, it is 78 necessary to understand the structure of the chitin wall and the mechanics of its formation. 79 E. histolytica does not encyst in an axenic culture, reptilian parasite Entamoeba invadens 80 has been opted as a model to study the encystation as it readily forms cyst when subjected to 81 starvation and osmotic stress (7). E. invadens occupy a similar host niche and create similar 82 colon pathology like E. histolytica (8). Also, E. invadens genome showed considerable sequence 83 similarity to that of E. histolytica (9). Most importantly, the cyst characteristics of E. histolytica 84 and E. invadens are quite similar; both formed a chitin walled tetranucleate cyst with a single 85 chromatoid body. A comparison of gene expression during encystation showed that they share a 5 86 large number of developmentally regulated genes. Encystation specific genes of E. invadens 87 were also found to over-expressed in clinical isolates of E. histolytica (10). Immunolocalization 88 experiments have shown that the cyst wall proteins identified in E. invadens like chitinase, Jacob 89 lectin, and Jessie are also present in the E. histolytica cysts isolated from patients (11). 90 Unlike the walls of fungi that contain multiple layers and many sugar polymers, the 91 Entamoeba cyst wall is single-layered and contains a single sugar polymer, chitin, forming 92 nearly 25% of its dry weight (12). The cyst wall also contains lectins with multiple chitin-93 binding domains like Jacob, Jessie, and chitinase (13). The assembly of different components 94 into the cyst wall is explained by the "wattle and daub model" (14). According to this model, 95 during the foundation stage, the Jacob lectin is secreted and binds to the constitutively expressed 96 Gal/GalNAc lectins on the cell membrane. During the "wattle" stage, chitin synthases produce  (16). Finally, during the daub phase, the Jessie lectin, which has a single CBD and a self-101 aggregation inducing-C-terminal domain, binds to this framework completing the cyst wall and 102 make it impermeable. 103 According to the ''wattle and daub'' model, Jacob lectins are deposited onto the surface 104 in the foundation stage, which then cross-links chitin fibrils in the "wattle stage", and addition of 105 Jessie lectins occur after 36 hours of encystation in the "daub stage". However, the expression 106 profiles of cyst wall proteins showed that most of them were over-expressed between 8 and 24 107 hours (17,18). In order to understand how different components were added to the wall, the 108 intermediate stages of cyst wall formation were investigated. As the in vitro encystation is an 6 109 asynchronous process, and so it is difficult to follow its progression. So we studied the 110 deposition of chitin by staining the cells with chitin-binding dyes and analyzed its maturity based 111 on detergent resistance. Localizing the chitin allowed us to follow the highly asynchronous 112 encystation as chitin is the most important maker of the encystation. We also studied the cyst 113 wall deposition with respect to the appearance of other cyst characteristics like chromatoid body 114 formation and nuclear division.

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The relationship between the actin cytoskeleton and chitin wall formation is well 116 established in fungi. Actin was found at sites where the cell wall synthesis is active like the bud, 117 growing apices, and sites of septum formation (19). In regenerating protoplasts of the 118 Saccharomyces cerevisiae, actin patches were found over the surface on which a new cell wall only when the cyst wall formation is complete. The wall forming early stages are not resistant to 132 detergent as staining with calcofluor white (CFW), the chitin-binding dye showed that the 133 numbers of detergent-resistant cells were less than the total number of CFW positive encysting 134 cells, especially in the early hours of encystations ( Fig 1A). In order to identify chitin positive 135 but detergent sensitive cells, encystation culture was examined at different time points (0 to 48 136 hr) by staining the chitin wall with CFW ( Fig 1B). No fluorescence was observed on the 137 encysting cells till the 9 th hour, but many cells with intense CFW fluorescence spots or cells with 138 fluorescence covering only a portion of their surface were observed between 12 to 16 th hour's 139 encystation culture, indicating the initiation of partial cyst wall formation. By 24 to 48 th hours,  to the self-assembly of wall components from a single point. Also, in a few encysting cells, the 159 chitin wall seemed to start from multiple points and then spread all over the surface, finally 160 overlapping and forming a continuous cyst wall ( Fig 2B) (14).  which were then used for immunofluorescence microscopy (Fig 4 A). EiCHS1 was not observed 177 in the trophozoite stage, but by 9 th hour EiCHS1 was localized in the cell distributed throughout 178 the cytoplasm (Fig 4 Aa). As the encystations progressed, by 12-16 hours, EiCHS1 was found to 179 be concentrated at one site (Fig 4 Ab), which was found to be the starting point for chitin In this study, we have revisited cyst wall formation and noticed the same sequence of 210 events during cyst wall formation as refer to Wattle and daub model (14). Here, we followed 211 encystation by co-localizing known cyst wall proteins like Jacob, Jessie, and chitinase using their  In contrary to the previously described model, we have noticed that Jessie expresses at 218 about 9 th hour of encystation and also found to co-localize with the growing chitin wall (Fig 6B).

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The early expression of Jessie also is in consensus with the expression profile of different cyst 11 220 wall proteins, including Jessie (17, 18). However, the expression of Jessie is relatively low and 221 hard to detect unless it is co-localized with chitin. We believe that in all the previous reports of 222 cyst wall biosynthesis, partial cyst wall formation was overlooked, and so the expression of 223 Jessie during the early hour of encystations. This is the first report where all the cyst wall 224 proteins (CWP) were co-localized with cyst wall chitin, and that helped us to spot the CWP 225 clearly.

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Our observations clearly indicate that the Jacob and chitin synthase deposition on the cell 227 surface are chitin independent, whereas the deposition of chitinase or Jessie is chitin dependent.  Encystation took place only inside the multicellular aggregates found in the encystation 235 culture, and it could be possible that the components of the cyst wall are secreted into the 236 intercellular space where they underwent self-assembly into complete chitin wall on the cell 237 surface from a starting point (Fig 7). Jessie lectin has a unique C-terminal domain that appears to 238 promote self-aggregation (14). Also, the ionic attraction between negatively charged Jacob, 239 Jessie, and chitinase and positively charged chitosan could be mediating such self-assembly. This have formed an aggregate in the cytoplasm (Fig 10A). When added to the encystation culture, 283 BDM dose-dependently inhibited the encystation, with 20 mM BDM reducing the encystation 284 efficiency to < 5% (Fig 10B). In the encystation culture, the cysts are formed only inside the 285 galactose ligand-mediated cell aggregates formed during encystation, and cell signaling required 286 for encystation takes place in these aggregates (47). The addition of BDM did not inhibit the cell 287 aggregates, but only a few chitin walled cysts were found in these aggregates, as shown by CFW 14 288 staining ( Fig 10C). However, in BDM treated cells, no change in the expression level of cyst 289 wall proteins was observed by RT-PCR ( Fig 10D) and CFW staining showed many cells with 290 disordered and patchy localization of chitin on their surface (Fig 10E). Further staining of BDM 291 treated cells for the nucleus, and chromatoid body revealed that most cells contained the 292 chromatoid body and four nuclei indicating the cells entered the encystation process and formed 293 mature cysts (Fig 10F). The presence of many tetranucleate cells with the chromatoid body BDM treated encystation culture contained both "wall-less cysts" and cysts with 302 abnormal walls. Instead of forming a uniform chitin wall (Fig 11A a), in BDM treated cells, 303 chitin was deposited in an irregular fashion (Fig 11A b), but in most cases, it was found to be 304 accumulated on one side (Fig 11A c, d). To find how the cortical actin disruption affects the cyst 305 wall formation, the cyst wall proteins were localized (Fig 11B). Immunostaining of Jacob in 306 BDM treated cells showed no change in its localization, it was present on the cyst surface in both 307 ''wall-less cysts'' and cyst with aberrant walls, just like in the normal cysts (Fig 11B, Jacob).

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Immunolocalization of chitin synthase (EiCHS1) in BDM treated cells obtained from 72-320 hour encystation culture showed that it was present on the surface of most cells, both cysts with 321 aberrant chitin walls (Fig 12 A, B) and the wall-less cysts (Fig 12 C, D). Thus the effects of     The PCR products were analyzed by running in a 1.5% agarose gel. EiARF was used as

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Secreted chitinases and Jessie binds to this framework. Cyst wall grows from these points (d) and 659 finally covers the whole surface (f). The major pathway is a-b-c-d-f and minor pathway is a-b-e-f.