Abstract
Vibrio cholerae is the causative agent of cholera, a form of diarrhoea, which continues to rage and remains a major public health problem in the developing world. The organism has the capacity to survive in diverse estuarine environments, as well as in the human host. Recent studies have suggested that interaction with a freshwater amoeba, Acanthamoeba castellanii, could be one possible mode of survival in the aquatic environment (Abd et al., 2005; Thom et al., 1992). It was also shown that V. cholerae could replicate intracellularly in A. castellanii. This prompted us to study its interaction with a parasitic amoeba, Entamoeba histolytica.
E. histolytica, the causative agent of amoebic colitis and amoebic liver abscess, is the second most common cause of death from parasitic disease worldwide (Stanley, 2003). In their natural environment, trophozoites of E. histolytica live in the colon of the human intestine together with the resident microflora, which under normal conditions is usually composed of a complex mixture of mainly anaerobic or microaerophilic bacteria (Mirelman, 1987). In order to examine the interaction of V. cholerae with the trophozoites of E. histolytica, a gentamicin assay was employed, as described previously (Venkataraman et al., 1997). In this assay, E. histolytica HM-1 : IMSS trophozoites (a kind gift from Professor Sudha Bhattacharyya, JawaharLal Nehru University, New Delhi, India) were suspended in serum-free TYI-S-33 medium at a concentration of 105 amoebae ml1. Trophozoites were incubated in triplicate in 24-well tissue culture plates (Falcon). Subsequently, V. cholerae O139, strain SG24 (a kind gift from Dr Ranjan Nandy, National Institute of Cholera and Enteric Research, Calcutta, India), was added to a final concentration of 107 cells ml1. The samples were incubated at 36 °C for 1 h, followed by the addition of 200 µg gentamicin ml1 for 2 h to kill the extracellular bacteria. After gentamicin treatment, the trophozoites were washed three times with PBS by centrifuging at 280 g for 7 min. After washing, trophozoites were counted with trypan blue to check viability and lysed by syringe passage in the presence of 0.01 % Triton X-100. Dilutions were plated on LuriaBertani agar plates for colony enumeration, which was found to be 5.53x103±0.18x103. The data suggested the presence of intracellular bacteria within the trophozoites of E. histolytica, with the trophozoites providing a protective barrier for V. cholerae against the effect of gentamicin.
To investigate the intracellular localization of the bacteria further, we carried out transmission electron microscopy of infected trophozoites. Microscopic pictures of trophozoites 1 h after infection revealed that V. cholerae O139 cells were localized intracellularly in the vacuoles (Fig. 1).