Interaction of Acanthamoeba and Shigella in vitro co-culture

[byungin]

Interaction of Acanthamoeba and Shigella in vitro co-culture

Jeong HJ, Jang ES, Han BI, Lee KH, Ock MS, Kong HH, Chung DI, Seol SY, Cho DT, Yu HS. Acanthamoeba: could it be an environmental host of Shigella? Exp Parasitol. 2007 Feb;115(2):181-6. Epub 2006 Sep 15.

CONTENTS

Ⅰ. INTRODUCTION-------------------------------------------------------------------------------- 1

Ⅱ. MATERIALS AND METHODS--------------------------------------------------------------- 4

1. Bacterial strains------------------------------------------------------------------------------- 4

2. Cell lines and culture conditions----------------------------------------------------------- 4

3. Virulence eval‎uation of bacterial strains-------------------------------------------------- 6

4. Intracellular bacterial growth in Acanthamoebae---------------------------------------- 6

5. Viability assay-------------------------------------------------------------------------------- 8

6. Transmission electron microscopy--------------------------------------------------------- 8

Ⅲ. RESULTS------------------------------------------------------------------------------------ 9

1. Plaque formation test of virulence and non-virulence Shigella sonnei---------------- 9

2. Uptake rates of three Acanthamoeba against two Shigella sonnei strains------------11

3. Changes of uptake rates by incubation temperature-------------------------------------13

4. Change uptake rates by culture media-----------------------------------------------------14

5. Viability of Acanthamoeba infected with virulence and non-virulence Shigella----15

6. Localization of Shigella in Acanthamoeba-----------------------------------------------16

Ⅳ. DISCUSSION------------------------------------------------------------------------------------ 21

Ⅴ. REFERENCES----------------------------------------------------------------------------------- 26

Ⅵ. ABSTRACT-------------------------------------------------------------------------------------- 31

FIGURES AND TABLE

Figure 1. Cytotoxicity test of HeLa cell by Shigella sonnei isolates----------------------------10

Figure 2. Uptake rates of three Acanthamoeba species. Bacteria uptake easily Acanthamoeba castellanii Neff strain assigned morphological group II-------------------------------12

Figure 3. Changes of uptake rates of  A. castellanii Neff by incubated temperature---------13

Figure 4. Changes of uptake rates of  A. castellanii Neff by type of medium----------------15

Figure 5. Detached cell number of A. castellanii Neff by two S. sonnies-----------------------17

Figure 6. Transmission electron microscopy shows morphological changes of Acanthamoeba intracellular organelle by Shigella infection--------------------------------------------18

Figure 7. Transmission electron microscopy shows Shigella attached to membrane of Acanthamoeba after 1 hour co-culture (A and B) ------------------------------------19

Figure 8. Shigella was engulfed by Acanthamoeba after 4 hours post co-cultures -----------20

Table 1. The list of Acanthameoba species in this study----

INTRODUCTION

Free-living amoebae and bacteria are involved in complex interactions, and it is known that amoebae act as environmental hosts of several intracellular pathogens, such as Legionella, Chlamydia, Mycobacterium, and Listeria spp. (Amann et al. 1997, Ly et al. 1990, Neumeister et al., 1997, Steinert et al. 1998, Winiecka-Krusnell and Linder, 2001). Interestingly, gene functions required by Legionella spp. for infection of protozoa are also required for infection of mammalian cells (Cianciotto et al. 1992, Gao et al. 1997, Hales and Shuman 1999, Segal and Shuman, 1999). It has even been suggested that growth in an amoebic intracellular environment might assist bacteria in their adaptation to mammalian phagocytic cells (Harb and Kwaik, 2000). Moreover, incorporation of bacteria into amoebic cysts has been shown to confer resistance to adverse environmental conditions such as exposure to biocidal agents (Barker and Brown, 1994).

Bacillary dysentery caused by Shigella species is an important cause of acute diarrheal disease in both developing and industrialized countries (Vila et al. 1994). Shigellosis is a serious public health problem in Korea, because large outbreaks of S. sonnei infections in many parts of this country during the period 1998 to 2000 were reported. The annual incidence of shigellosis was estimated at about 10 cases before 1997 but explosively increased to about 1,000 to 2,500 cases during the period 1998 to 2000 (NIH of Korea report, 2002). But epidemiological features of shigellosis were not known exactly. What could transmit Shigella (that can infect only primate included human) to food, drink or other geographical area?

Free-living amoebae, such as Acanthamoeba spp., are commonly found in natural aquatic systems and in soil (Martinez and Visvesvara, 1997), and even within the intestines of humans (Thamprasert et al. 1993, Zaman et al. 1999) and reptiles (Sesma and Ramos, 1989); hence, they are expected to encounter and ingest Shigella. Indeed, King et al. (1988) reported the survival of Salmonella enterica serovar Typhimurium within Acanthamoeba castellanii during chlorination, suggesting a protective intracellular habitat for the bacteria. Also, Escherichia coli O157 can survive in soil protozoan (Barker et al. 1999). The role of protozoa in the environmental survival of Shigella has not been studied. There is growing concern about the survival of pathogens in sewage sludges disposed of on land now that green laws limit sea dumping. Soils contaminated with organic matter and sewage waste contain greatly increased numbers of protozoa such as acanthamoebae (Rodriguez-Zaragoza, 1994; Sawyer, 1989). Thus it is highly likely that Shigella in soil and slurry will be preyed on by free-living amoebae which could be potential vectors for the spread of this pathogen. Using laboratory microcosms we have studied the potential for a ubiquitous free-living protozoan, Acanthamoeba to support the growth of Shigella.

In this study we describe the establishment of conditions for the growth and replication of Shigella in an amoebic intracellular environment, and we determine whether bacterial growth conditions affect the survival, replication, and cytotoxicity of Shigella within Acanthamoeba.

MATERIALS AND METHODS

Bacterial strains.

Two Shigella sonnei strains isolated from human infection in Korea. The 80DH248 strain was isolated at 1980 and has been lost their virulence ability by maintained in artificial medium and many times generation. The other strain 99OBS1 was isolated from sample of big outbreak shigellosis and has virulence ability.

Cell lines and culture conditions.

The three isolates of amoebae were obtained from the American Type Culture Collection (ATCC). They used in these experiments were Acanthamoeba castellanii Neff strain (ATCC #30010), A. astronyxis Ray & Hayes strain (ATCC #30137) and A. healyi OC-3A strain (ATCC #30866). Table 1 was shown characteristics of these isolates. These were maintained in an axenic culture medium, peptone-yeast extract-glucose (PYG) broth, in 25-cm³ tissue culture flasks incubated at 24°C, until near-confluence was reached. Amoebic suspension was examined by bright-field microscopy before use, and the number of cells was determined by cell counting in a Burker chamber. HeLa cells from the American Type Culture Collection were maintained in RPMI 1640 medium supplemented with L-glutamine (final concentration, 2 mM), HEPES (final concentration, 10 mM), fetal bovine serum (final concentration, 10% [vol/vol]), and gentamicin sulfate (final concentration, 10 µg/ml).

Table 1. The list of Acanthameoba species in this study

Strain	# ATCC	Environmental Source	species designation	Group 1)
Ray & Hayes Neff DC-3A	30137 30010 30866	Soil Soil GAE	A. astronyxis A. castellanii A. healyi	Group I Group II Group III

¹⁾ Grouping was according to method of Pussard and Pons (1970)

Virulence eval‎uation of bacterial strains.

Bacterial strains were grown either in LB or on LA with appropriate antibiotics. Shigellae grown to the late-logarithmic-growth phase in LB and added to culture flask of HeLa cell grown to the late-logarithmic-growth phase (No. of bacteria: No. of HeLa cell = 10: 1), and after the incubation at 37°C during 2 hr, washed three times with phosphate-buffered saline (PBS). Eval‎uation of virulence was determined by formed or not cell plaques.

Intracellular bacterial growth in Acanthamoeba.

Amoebae were grown in 5 ml of PYG medium. The tissue culture flask was gently shaken, and the PYG containing non-adherent amoebae was removed. New PYG was added, and the amoebae were taken off by incubation on ice for 30 min. The suspension was centrifuged for 5 min at 200 x g, and the pellet was washed with PBS and resuspended in PYG. The suspension was added to each well of a 6-well plate (10⁶ amoebae/well). Amoebae were then incubated for 24 hr at 30°C to allow them to adhere. The number of amoebae/well was calculated once more before infection. To infect amoebae, invasive or noninvasive bacterial inocula were diluted in PYG to give a final concentration of 20 to 100 bacteria per amoeba. The plate was gently centrifuged (for 5 min at 500 x g) in order to promote contact between bacteria and amoebae and was then incubated for 60 min at 37°C. After the incubation, the medium was changed to PYG supplemented with gentamicin sulfate (final concentration, 50 µg/ml) in order to kill extracellular bacteria. After incubation for 60 min at 37°C, the medium was changed to maintenance medium, PYG supplemented with gentamicin at a final concentration of 10 µg/ml, for continuing incubation. The cells were washed twice with PBS and lysed in 0.5% sodium deoxycholate (Johansson et al., 2003), and the number of CFU was determined by plating samples on LA plates.

Viability assay

The viability of host cells was determined at 16 hr post-infection by using trypan blue, followed by quantification of stained cells and total cells by use of a Burker chamber.

Transmission electron microscopy.

Acanthamoeba was infected with Shigella sonnei as described above. At 16 h postinfection, the cells were washed twice with PBS and fixed with 50% sodium cacodylate (pH 7.4), 1% glutaraldehyde, and 3% paraformaldehyde for 1 h at 4°C. Cells were harvested by pipetting up and down, pelleted for 5 min at 600 x g and 4°C, and resuspended in fixation buffer diluted 1:10. Transmission electron microscopy was performed using standard procedures.

RESULTS

Plaque formation test of virulence and non-virulence Shigella sonnei

We initiated our experiments by HeLa cell plaque forming test by Shigella sonnei 99OBS1 and 80DH248 strains. Two hours after co-culture, incubated HeLa cell without Shigella in cell culture medium for overnight. HeLa cell co-incubated with Shigella sonnei 80DH248 strain were maintained intact morphology (Figure 1A & 1B). But Shigella sonnei 99OBS1 strain infected in HeLa cell and induced cell aggregation (initiated about 18 hour after co-culture, Figure 1C & 1D). After 24 hour co-culture, Most of aggregated cells were detected from cell culture plate and many plaques were formed (Figure 1E & 1F). The size and number of plaques were increased in time.

Figure 1. Cytotoxicity test of HeLa cell by Shigella sonnei isolates.

Panel A & B; 80DH248 (A; ×200, B; ×400). Panel C, D, E, F; 99OBS1, (C; ×100, 18 hr incubation, D; ×400, 18 hr incubation, E; ×200, 24 hr incubation, F; ×400, 24 hr incubation).

 Uptake rates of three Acanthamoeba against two Shigella sonnei strains

We eval‎uated efficiency uptake rates of invasive and non invasive Shigella sonnei strains in three Acanthamoeba species, A. castellanii Neff (isolated from soil), A. astronyxis Ray & Hayes (isolated from soil), and A. healyi OC-3A (isolated from human brain) (Figure 2). The number of viable intracellular bacteria was determined as a percentage of the initial inoculum (for details, see Materials and Methods). A .castellanii Neff strain was infected with the number of Shigella sonnei 99OBS1, 80DH248 and maintained for a long time in their cytoplasms than other Acanthamoeba species. Uptake rates of Neff strain against Shigella sonnei 99OBS1 and 80DH248 were 59.42% and 24.09%, respectively. But we observed a significant decrease in the numbers of viable bacteria recovered from infected A. asronyxis Ray & Hayes and A. healyi OC-3A. The uptake rates of Ray & Hayes against 99OBS1 and 80DH248 were 0.55% and 0.13%, respectively. Those of OC-3A were estimated by 0.74% and 0.52%, respectively. Shigella sonnei 99OBS1 strain (virulence strain) was recovered in higher numbers than non-virulence strain Shigella sonnei 80DH248 strain in all experiment.

(%)

Figure 2. Uptake rates of three Acanthamoeba species. Bacteria uptake easily Acanthamoeba castellanii Neff strain assigned morphological group II

Changes of uptake rates by incubation temperature

Three temperature points of incubation were eval‎uated as the most easily uptake of Shigella sonnei to Acanthamoeba. The uptake rate of Neff post co-culture incubated at 18°C was increased in time to 6 hours, but after 6 hours uptake rate was decreased in time (Figure 3). One, 2.5, 4, 6 hour incubated at 18°C after co-culture, the uptake rates of A. castellanii Neff were 10.53%, 27.25%, 59.43%, 61.75%, respectively. Of other incubation temperature, uptake rates of Neff were below 1% in all of check time point.

Figure 3. Changes of uptake rates of A. castellanii Neff by incubated temperature.

Change uptake rates by culture media

To eval‎uate of uptake rate of Acanthamoeba by change culture medium, we used original culture medium PYG (media) and free-media (proteose peptone, yeast extract, glucose exclude in PYG medium) (Figure 4). The uptake rates of Neff cultured in free-media were higher (about above 10 fold) than those cultured in media in all check time points. Four hours after co-culture, the uptake rate of Neff cultured in free media and media was highest. Uptake rates of Neff cultured in free media 1, 2.5, 4, 6 hours after co-culture were 10.53%, 27.25%, 61.75%, 59.43%, respectively. Uptake rates of Neff cultured in PYG media 1, 2.5, 4, 6 hours after co-culture were 0.58%, 4.26%, 4.5%, 2.01%, respectively.

Figure 4. Changes of uptake rates of A. castellanii Neff by type of medium.

Viability of Acanthamoeba infected with virulence and non-virulence Shigella.

Under phase-contrast microscopy, A. castellanii Neff infected with invasive wild-type S. sonnei 99OBS1 displayed a significant loss of adherent cells over 6 h of infection (Fig. 5). A similar phenomenon is observed when human monocytic cells are infected with salmonellae: infected cells become detached, whereupon most of the viable intracellular bacteria can be recovered from detached cells (Libby, et al. 2000). Therefore, the numbers of viable bacteria recovered from attached and detached Neff at 6 h postinfection were determined by using LB-grown cultures of the bacteria. The results show that the number of viable Shigella bacteria recovered from the detached amoebae was higher than that recovered from the attached fraction (data not shown). We then reasoned that detachment of Neff after infection with S. sonnei could be the result of a cytotoxic effect mediated by the infecting bacteria. Although the number of the detached cell was smaller than virulence strain 99OBS1, non-virulence S. sonnei 80DH248 also could detach amoeba from the culture flask.

Localization of Shigella in Acanthamoeba

Beside, Shigella non-infected Acanthamoeba have their intact membrane and cell organelle (Figure 6A& B), cell structure and cellular organelle of Shigella infected Acanthamoeba were ruptured and changed their morphology (Figure 6C & D). These phenomenons might be caused by Shigella toxin and contact. Invasive S. sonnei 99OBS1 was localized with intact their membrane in vacuole of Acanthamoeba (Figure 6, 7 and 8).

Figure 5. Detached cell number of A. castellanii Neff by two S. sonnei s

Figure 6. Transmission electron microscopy shows morphological changes of Acanthamoeba intracellular organelle by Shigella infection. Non-infected amoeba (A and B), After 4hr postinfection with S. sonnei 99OBS1 (C and D). (A) Magnification ´4,000; bar, 2 mm. (B) Magnification, ´20,000; bar, 200 nm. (C) Magnification, ´5,000; bar, 1 mm. (D) Magnification, ´15,000; bar, 500 nm.

Figure 7. Transmission electron microscopy shows Shigella attached to membrane of Acanthamoeba after 1 hour co-culture (A and B). Intra localization of S. sonnei in Acanthamoeba after 4 hours co-cultures (C and D). (A) Magnification ´8,000; bar, 1 mm. (B) Magnification, ´15,000; bar, 500 nm. (C) Magnification, ´5,000; bar, 1 mm. (D) Magnification, ´10,000; bar, 500 nm.

Figure 8. Shigella was engulfed by Acanthamoeba after 4 hours post co-cultures (A and B). Multiple shigella localized in vacuoles of Acanthamoeba after 4 hours post co-culture (C and D), (A) Magnification ´5,000; bar, 1 mm. (B) Magnification, ´15,000; bar, 500 nm. (C) Magnification, ´8,000; bar, 1 mm. (D) Magnification, ´12,000; bar, 500 nm.

DISCUSSION

Free-living amoebae are gaining increasing attention as ubiquitous eukaryotes influencing our perception of human bacterial pathogens in the environment. For example, the facultatively intracellular mammalian pathogens Legionella pneumophila and mycobacteria are also known to prosper within free-living amoebae, implying that such bacteria are adapted to an intracellular environment in a more general sense (Neumeister et al, 1997; Steinert et al., 1998). Furthermore, L. pneumophila is known to use the same sets of genes for multiplying in human macrophages and A. castellanii (Harb and Kwaik, 2000).

Shigellae are rather promiscuous in their ability to invade and replicate in mammalian host cells. In contrast, the interaction between shigellae and Acanthamoeba species has not been described in any detail. In the present study we observed a preferential uptake of virulence and non-virulence Shigella sonnei strains in three isolates of Acanthamoeba tested. Virulence strain was more efficiently invasive in all Acanthamoeba isolates tested. These results might be 80DH248 strain (non-virulence strain) have deletion mutation in their virulence gene, virG (unpublished data). The role of virG protein is actin polymerization of Shigella one side polar in the host cell cytoplasm using host mono form actin. Because, S. sonnei 80DH248 could not actin polymerization in Acanthamoeba, so number of uptake into Acanthamoeba was smaller than those of virulence strain 99OBS1 (Figure 1).

Uptake rates of A. castellanii Neff strain were highest among other Acanthamoeba. Up to now, morphological group II Acanthamoeba only reported that included in endosymbionts in their cytoplasm. Although could not know about exact reason, this phenomenon was gave rise to by their genetic or epidemiologic abilities. Group II Acanthamoeba was the most isolated from our environment, especially water related environment. Bacteria could be survived in only water system or water related systems. Thus group II Acanthamoeba and bacteria in water could easily contact, their uptake ability might have been increased during long time.

The incubation temperature was very important elements among efficient bacteria uptake system of Acanthamoeba. Temperature of incubation affected uptake rates of Acanthamoeba up to above 100 folds (Figure 3). Virulence genes of Shigella could be activated at 37°C, so incubation temperature the more close to 37°C, the more activate virulence genes and then Shigella could kill amoeba. The temperature of our soil might be below 24°C, the efficiency of uptake rate in environment might be similar to result of Acanthamoeba incubated at 18°C.

Shigella more effetely uptake in amoeba incubated free medium than PYG medium. This results shows that S. sonnei multiply in Acanthamoeba was affected by medium. The environment situation was similar to free medium system; uptake of Shigella by Acathamoeba might be existence.

Prolonged incubation of the bacterial-amoebic cultures resulted in a gradual change in morphology and eventually in the disappearance of the host cells (Fig. 6C & D). A related phenomenon has been reported by Ly and Müller (Ly and Muller, 1990) for cocultures of Listeria monocytogenes and Acanthamoeba species. Listeriae were taken up by the amoebae, and the bacteria replicated intracellularly. However, longer incubations led to release of bacteria and encystment, with the intracellular listeriae losing viability in cysts.

A portion of the observations implied that the loss of shigellae in amoeba was not simply a reflection of intracellular killing of the bacteria. Shigella, like several other bacterial pathogens, including Mycobacterium, Legionella, and Brucella spp., replicate within membrane-bound vacuoles inside macrophages. While we did not attempt to define the nature of intracellular vesicles, electron microscopy of Salmonella-infected A. rhysodes showed that the bacteria were localized within membrane-bound vacuoles (Figure 7 through 8).

Free-living amoebae can harbor bacteria inside their cysts, giving them a microhabitat protecting them from environmental hazards. Furthermore, a study by King et al. (26) showed that the bacterium-protozoan association provides increased numbers of bacteria with increased resistance to free chlorine residuals, which can lead to the persistence of bacteria in chlorine-treated water. It has also been reported that amoeba-grown L. pneumophila displays increased intracellular survival and replication in macrophages (Cirillo et al., 1998) and that intracellular growth in A. castellanii affects the monocyte entry mechanism and enhances the virulence of L. pneumophila. Although the present study included only a restricted set of shigellae and acanthamoebae, and therefore formally may not reflect all possible forms of interactions, our results show that Acanthameoba was able to ingest shigellae and that subsequent events included intracellular bacterial localization. It thus remains possible that free-living amoebae function as environmental hosts for shigellae and those amoebae may participate in the transmission of outbreaks of shigellosis in Korea.

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가시아메바와 이질균의 같이배양 (co-culture)에 의한 상호관계 규명

한 병 인

부산대학교 대학원 의학과

Abstract

1998년부터 국내에 세균성이질의 발생이 여러 지역에서 보고되고 있어 공중보건학상 매우 큰 문제가 되고 있다. 그러나 이러한 세균성이질의 역학에 대한 연구는 잘 이루어 지고 있지 않고 있다. 이번 연구에서 이질균의 가시아메바내에서의 탐식, 증식 및 세포독성에 대한 연구를 하였다. 병원성, 비병원성 이질균 2분리주와 3 종의 가시아메바 (A. castellanii Neff주, A. astronyxis Ray & Hayes주, and A. healyi OC-3A주)를 사용하여 실험하였다. 이 중 Neff 주의 경우 다른 가시아메바들 보다 많은 숫자의 이질균을 탐식하였고 또한 오랜 시간 동안 세포내에 가지고 있었다.  병원성 세균성 이질균 99OBS1주 는 비병원성 80DH248균주 보다 매 실험에서 많은 수가 관찰되었다. 온도별로 탐식률을 조사한 결과 18°C에서 가장 높았고 6시간째 가장 높았고 그 이후 점차 감소하였다. 배지에 따른 탐식률은 영양분이 없는 free-media에서 가시아메바 배양 배지 PYG보다 높은 탐식율을 보였다.  이 중 Neff 주는 free-media배지내 에서 PYG 배지 내에서 보다 10배 이상 높은 탐식률이 관찰되었다. 이질균은 가시아메바의 소포체내에서 관찰되었다. 결론적으로 자유생활아메바는 이질균의 환경내의 숙주역할을 할 수 있으며 아메바가 국내 이질 발생에 영향을 끼칠 것으로 생각한다.