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In vivo killing of Giardia trophozoites harbouring bacterial endosymbionts by intestinal Paneth cells: an ultrastructural study

Published online by Cambridge University Press:  19 October 2004

K. A. EL-SHEWY  and
R. A. EID
K. A. EL-SHEWY
Affiliation:
Microbiology and Parasitology Department, Faculty of Medicine, Suez Canal University, Ismailia, Egypt
R. A. EID
Affiliation:
Electron Microscopy Center, Faculty of Sciences, Zagazig University, Egypt
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Abstract

To date Paneth cells have not previously been reported to kill Giardia trophozoites and other protozoa in vivo. Here we report the first evidence for in vivo killing of Giardia trophozoites by intestinal Paneth cells. Transmission electron microscopic (TEM) examination of duodenal specimens taken from naturally infected mice revealed that only Giardia trophozoites harbouring peripheral bacterial endosymbionts were destroyed and lysed in the vicinity of the activated Paneth cells. Additionally, intestinal epithelium was more affected by Giardia harbouring bacterial endosymbionts than Giardia with no endosymbionts. Our findings imply that the bacterial endosymbionts within Giardia trophozoites have a role in both host protective and pathological mechanisms, probably through altering the trophozoite antigencity. These observations might shed light on the diversity in infectivity and host specificity of Giardia species.

Keywords

endosymbiosisgiardiasiselectron microscopyPaneth cellsin vivo killingnaturally infected mice
Type
Research Article
Information
Parasitology , Volume 130 , Issue 3 , March 2005 , pp. 269 - 274
Copyright
2004 Cambridge University Press

INTRODUCTION

Paneth cells are epithelial granulocytes located at the base of the crypts of Lieberkühn in the small intestine of many mammalian species (Ouellette & Selsted, 1996). These cells contribute actively to mucosal immunity through secretion of antimicrobial polypeptides including α-defensins (cryptdin) that are released from the intestinal Paneth cells in response to bacteria and bacterial antigens, but not to protozoa. These antimicrobial polypeptides kill a wide range of organisms including bacteria, viruses, fungi and tumour cells (Martin, Ganz & Lehrer, 1995; Ayabe et al. 2000).

Some in vitro studies have reported that Giardia trophozoites were destroyed and lysed by defensins and other antimicrobial polypeptides derived from the intestinal Paneth cells (Aley et al. 1994). However, no evidence has been reported so far for the in vivo killing of Giardia trophozoites when these cells are stimulated.

Giardia trophozoites, in common with some other protozoan parasites, demonstrate bacterial endosymbiosis (de-Souza & Motta, 1999). The significance of the bacterial endosymbionts within Giardia is not well defined. It is possible that they could alter trophozoite pathogencity, metabolism, range of infectivity and host specificity, as they do in other protozoa (Nemanic et al. 1979).

In this report the ultrastructural interaction between Giardia trophozoites harbouring bacterial endosymbionts and mouse intestinal Paneth cells is described.

MATERIALS AND METHODS

Animals

Light and transmission electron microscopic (TEM) examinations were conducted for examination of the duodenal specimens taken from Giardia infected mice. Twenty-five laboratory reared albino mice (Tuck ordinary strain), naturally infected with Giardia muris as detected by screening of their faecal suspension (Roberts-Thomson, Stevens & Mahmmoud, 1976), were chosen. It was found that 10 of these 25 mice were heavily infected with G. muris trophozoites. These heavily infected mice were sacrificed.

Mice were dissected and duodenal segments were removed. Segments were fixed in alcoholic Bouin with 10% buffered neutral formalin for subsequent light microscopic examination. Similar segments were fixed in 2·5% glutaraldehyde for TEM examination.

Transmission electron microscopy

Specimens prepared for TEM were cut into 2–3 mm2 sections and immediately fixed in 2·5% glutaraldehyde with 0·1 M sodium cacodylate buffer (pH 7·2) at 4 °C for 2–3 h. Specimens were post-fixed in 1% osmium tetroxide, dehydrated in ascending grades of ethyl alcohol, and embedded in Spurr's resin. Sections (0·5 μm) were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Jeol 1200 EX TEM at 80 KV (Glauert, 1980).

RESULTS

TEM examination of the specimens taken from naturally infected mice demonstrated many Giardia trophozoites on the brush border, at the base of the crypts as well as among the intestinal microvilli. The internal fine structure of these trophozoites was clearly demonstrated (Fig. 1A, B). Severe blunting and atrophy of microvilli with occasional loss of basic epithelial morphology and distorted Goblet cells were observed in some of the examined duodenal sections (Fig. 1C, D).

Fig. 1. (A) Transmission electron micrograph of duodenal specimen taken from naturally infected mice demonstrating intestinal microvilli (Iv) covering the intestinal epithelium (Ie), with Giardia trophozoites (Gt) in the intestinal lumen (Lu). (×5000.) (B) Higher magnification of the Giardia trophozoite seen in (A) Adhesive disc (Ad), nucleus (N), peripheral vesicles (Pv), cell membrane (Cm), marginal groove (Mg), and the striated rim of the groove (Sr). Ax represents the axonemes of the flagellum, cytoplasmic tubules (Ct), intestinal microvilli (Iv), and lumen (Lu). (×17500.) (C) TEM of duodenal mucosa infected with Giardia trophozoites, demonstrating marked stimulation of the Goblet cell (Gc), with Goblet secretion (Gs), in between the intestinal epithelium (Ie) covered with microvilli (Iv). (×9500.) (D) Severely damaged epithelial mucosa as the result of Giardia infection including shortened microvilli (Sv), distorted microvilli (Dv), and marked mucosal atrophy in between the affected microvilli (arrow heads). Intestinal epithelium (Ie). (×20700.)

In these duodenal specimens bacterial endosymbionts within Giardia trophozoites were demonstrated. Bacteria were found in close contact with the outer surface of some Giardia trophozoites. In some others, they were inundating the trophozoite cell membrane. Electron-lucent spaces were detected surrounding the bacterial endosymbionts and bounded with aggregation of cytoplasmic granules (Fig. 2A, B). No evidence was found for digestion of these bacterial endosymbionts by Giardia trophozoites. There were, however, many electron-lucent vacuoles forming a track directed towards the periphery of the trophozoite. Endosymbionts were concentrated peripherally in direct contact with the intestinal microvilli, and were uncommon in the nuclear area (Fig. 2C).

Fig. 2. (A) TEM picture of duodenal mucosa showing the first step in the bacterial endosymbiosis. B represents bacterial body in between the intestinal microvilli (Iv) and Giardia trophozoite (Gt). The bacterial body is pushing itself toward the cell membrane (Cm) of the trophozoite by making invagination in it (arrow heads). Adhesive disc (Ad). (×27500.) (B) Higher magnification of the Giardia trophozoite (Gt), with endosymbiosis, showing multiple bacterial endosymbionts (Be) completely invaginating inside the cell membrane of the Giardia trophozoite (arrows), and bounded with electron-lucent space (Es), and surrounded with many cytoplasmic granules (star). Note the peripherally location of endosymbionts. Iv, intestinal microvilli. (×71400.) (C) Longitudinal section of Giardia trophozoite showing multiple bacterial endosymbionts (Be), with a track of electro-lucent vacuoles directed towards the periphery of the trophozoites (V). Cytoplasmic granules (stars); Ax, represents axonemes of the flagellum (F); intestinal villi (Iv); Paneth cell (Pc). (×20000.)

No gross morphological changes were detected in the Giardia trophozoites as the result of these bacterial endosymbionts. However, Giardia trophozoites were found completely lysed at the base of the duodenal crypts and close to the Paneth cells (Fig. 3A–D).

Fig. 3. (A) TEM picture of a transverse section in an intestinal crypt showing ultrastructural interactions between Giardia trophozoite, bacterial endosymbionts, and Paneth cells. Cell membrane of Giardia trophozoite (Gt) appears lysed at the dorsal surface of the parasite (black arrows) with peripheral bacterial endosymbionts (Be) in close contact with Paneth cells (Pc). The interface between the trophozoite and the cells is filled with a dense secretion (S). Paneth cells appear active with many secretory granules inside (G), granular cytoplasm (stars), damaged mucosa and severely distorted epithelial microvilli (white arrows); Lu, lumen. (×17500.) (B–D) Transverse sections of Giardia trophozoite (Gt) at the base of the intestinal crypt demonstrating different stages of lysis and destruction of the Giardia body by the surrounding Paneth cells (Pc). B, bacteria; lysed cell membrane (arrows); flagellum (F); atrophied mucosal lining (arrow head), with lysed trophozoites in between the microvilli (double arrows). (×14200 (B), 13600 (C) and 20000 (D).) (E) TEM picture demonstrating Giardia trophozoites (Gt) lodged in between the intestinal microvilli (Iv). Note that the Giardia trophozoite is free from bacterial endosymbionts and the surrounding Paneth cells (Pc) appear inactive and not stimulated. Inactive Goblet cells (Gc). (×6600.)

Paneth cells were found to be highly active with many secretory granules inside, especially those in contact with Giardia trophozoites harbouring peripheral endosymbionts. On the other hand Paneth and Goblet cells associated with Giardia trophozoites and having no endosymbionts were found inactive and the trophozoites were demonstrated to be completely intact and discrete. Mucosal lining in close contact with such trophozoites was observed to be almost intact compared to the severely damaged mucosa observed with trophozoites containing endosymbionts (Figs 1D and 3E).

DISCUSSION

The precise nature of the interaction between Giardia and its host remains conjectural because of the paucity of published studies. The present ultrastructural report describes an interaction between Giardia trophozoites and intestinal Paneth cells in the presence of bacterial endosymbionts.

Although intestinal Paneth cells secrete antimicrobial peptides that affect many intestinal pathogens, Giardia as many other protozoan parasites have not previously been reported to be killed by Paneth cells or by their antimicrobial secretions (Jones & Bevins, 1992; Selsted et al. 1992; Aley et al. 1994).

In the present report we observed lysis of the cell membrane of Giardia trophozoites when bacterial endosymbionts came in close contact with Paneth cells. Intestinal Paneth cells associated with such trophozoites were found highly activated and with many internal secretory granules.

We could not detect lysis of any Giardia trophozoites free from bacterial endosymbionts and we therefore speculate that the bacterial endosymbionts stimulate Paneth cells. Giardia trophozoites did not stimulate Paneth cells when these cells were exposed to trophozoites in vitro (Ayabe et al. 2000). However, the authors did not exclude the possibility that these trophozoites and other eukaryotes may induce Paneth cell secretion by an unidentified mechanism in vivo.

The reported activation and lytic effect of Paneth cells were seen in all duodenal specimens examined with trophozoites harbouring bacterial endosymbionts. This lead to our proposal that bacterial endosymbionts activate Paneth cells, resulting in release of their lytic peptides mainly defensins. It is likely that defensins are responsible for Giardia lysis since they account for 70% of the released bactericidal activity of Paneth cells (Ayabe et al. 2000). In an earlier study, α-defensins demonstrated a remarkable lysis and killing of Giardia trophozoites in vitro (Aley et al. 1994).

The pathological changes observed in this study as the result of Giardia trophozoites indicated that intestinal epithelium was more affected by Giardia with bacterial endosymbionts than those without endosymbionts suggesting that bacterial endosymbionts might alter the trophozoite pathogenicity.

The reported variation in the pathogenicity and the stimulation of the Paneth cells to react actively with the trophozoites could be due to variation in the trophozoite surface antigens and consequently the immunopathological process (Meng, Hetsko & Gillin, 1993). Such variation in the Giardia surface antigens as the result of bacterial endosymbionts has been proposed earlier (Meyer, 1990).

Bacterial flora and other organisms that live in the intestine appear to provide the several necessary services for healthy development and the suggested functions of the bacterial endosymbionts described here could explain the significance of endosymbiosis in Giardia and other similar protozoa.

The authors would like to thank Dr Samir A. Zaki, Associate Professor of histology, Faculty of Medicine, Zagazig University, Egypt, for his kind assistance in this work.

References

REFERENCES

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Figure 0

Fig. 1. (A) Transmission electron micrograph of duodenal specimen taken from naturally infected mice demonstrating intestinal microvilli (Iv) covering the intestinal epithelium (Ie), with Giardia trophozoites (Gt) in the intestinal lumen (Lu). (×5000.) (B) Higher magnification of the Giardia trophozoite seen in (A) Adhesive disc (Ad), nucleus (N), peripheral vesicles (Pv), cell membrane (Cm), marginal groove (Mg), and the striated rim of the groove (Sr). Ax represents the axonemes of the flagellum, cytoplasmic tubules (Ct), intestinal microvilli (Iv), and lumen (Lu). (×17500.) (C) TEM of duodenal mucosa infected with Giardia trophozoites, demonstrating marked stimulation of the Goblet cell (Gc), with Goblet secretion (Gs), in between the intestinal epithelium (Ie) covered with microvilli (Iv). (×9500.) (D) Severely damaged epithelial mucosa as the result of Giardia infection including shortened microvilli (Sv), distorted microvilli (Dv), and marked mucosal atrophy in between the affected microvilli (arrow heads). Intestinal epithelium (Ie). (×20700.)

Figure 1

Fig. 2. (A) TEM picture of duodenal mucosa showing the first step in the bacterial endosymbiosis. B represents bacterial body in between the intestinal microvilli (Iv) and Giardia trophozoite (Gt). The bacterial body is pushing itself toward the cell membrane (Cm) of the trophozoite by making invagination in it (arrow heads). Adhesive disc (Ad). (×27500.) (B) Higher magnification of the Giardia trophozoite (Gt), with endosymbiosis, showing multiple bacterial endosymbionts (Be) completely invaginating inside the cell membrane of the Giardia trophozoite (arrows), and bounded with electron-lucent space (Es), and surrounded with many cytoplasmic granules (star). Note the peripherally location of endosymbionts. Iv, intestinal microvilli. (×71400.) (C) Longitudinal section of Giardia trophozoite showing multiple bacterial endosymbionts (Be), with a track of electro-lucent vacuoles directed towards the periphery of the trophozoites (V). Cytoplasmic granules (stars); Ax, represents axonemes of the flagellum (F); intestinal villi (Iv); Paneth cell (Pc). (×20000.)

Figure 2

Fig. 3. (A) TEM picture of a transverse section in an intestinal crypt showing ultrastructural interactions between Giardia trophozoite, bacterial endosymbionts, and Paneth cells. Cell membrane of Giardia trophozoite (Gt) appears lysed at the dorsal surface of the parasite (black arrows) with peripheral bacterial endosymbionts (Be) in close contact with Paneth cells (Pc). The interface between the trophozoite and the cells is filled with a dense secretion (S). Paneth cells appear active with many secretory granules inside (G), granular cytoplasm (stars), damaged mucosa and severely distorted epithelial microvilli (white arrows); Lu, lumen. (×17500.) (B–D) Transverse sections of Giardia trophozoite (Gt) at the base of the intestinal crypt demonstrating different stages of lysis and destruction of the Giardia body by the surrounding Paneth cells (Pc). B, bacteria; lysed cell membrane (arrows); flagellum (F); atrophied mucosal lining (arrow head), with lysed trophozoites in between the microvilli (double arrows). (×14200 (B), 13600 (C) and 20000 (D).) (E) TEM picture demonstrating Giardia trophozoites (Gt) lodged in between the intestinal microvilli (Iv). Note that the Giardia trophozoite is free from bacterial endosymbionts and the surrounding Paneth cells (Pc) appear inactive and not stimulated. Inactive Goblet cells (Gc). (×6600.)