Crescent Bodies of Parachlamydia acanthamoeba and Its Life Cycle within Acanthamoeba polyphaga : an Electron Micrograph Study

Abstract

Parachlamydiaceae are endosymbionts of free-living amoeba first identified in 1997. Two developmental stages, elementary and reticulate bodies, were observed; however, their localization and proportions according to culture condition and duration remain unknown. The life cycle of Parachlamydia acanthamoeba within Acanthamoeba polyphaga was studied by transmission electron microscopy of 8-, 36-, and 144-h coculture. Morphometry and quantification were performed using SAMBA software. The elementary body, the predominant stage within the amoebae, was located mainly within their vacuoles. Legionella was the first pathogen demonstrated to multiply and persist in free-living amoebae (21), which are common inhabitants of natural aquatic environments and water systems (11,19). Since then, several other intracellular bacterial pathogens were shown to infect free-living amoebae, including Chlamydia pneumoniae (6), Mycobacterium avium (24), Listeria monocytogenes (13), an Ehrlichia-like organism (15), the Legionella-like amoebal pathogens (3, 9), Afipia species (12), and Chlamydia-like endosymbionts (1, 4). The latter, Parachlamydia acanthamoeba, could be pathogenic for humans (7a). However, there are only eight strains of Parachlamydiaceae described to date, including the BN9 endosymbiont (1) and Hall's coccus (4). Amann et al. demonstrated the presence of two developmental stages, a thin-walled gram-negative stage and a gram-positive infective stage (1). These two stages, i.e., reticulate and elementary bodies (well known for Chlamydia spp . [16,17]), were also subsequently reported by Horn et al. and by Fritsche et al. for additional Parachlamydiaceae strains (7, 10). However, the localization of these bodies within free-living amoebae according to culture condition and duration was not yet studied. It is unknown how they exit the protozoan host. Fritsche et al. showed the presence of Chlamydia-like inclusions in a cyst of Acanthamoeba (7), suggesting that amoebae could also act as Trojan horses for the Parachlamydiaceae, since amoebae are resistant to various disinfecting solutions (5,20,26) and to extreme temperature, pH, and osmolarity conditions while encysted (reviewed in reference 18).In the present paper, we report on a third and as yet unknown developmental stage, the crescent body, and on the life cycle of P. acanthamoeba within Acanthamoeba polyphaga. Strains, culture, and fixation. P. acanthamoeba strain BN9 (ATCC VR 1471) was kindly provided by R. Amann. Parachlamydia sp. "Hall's coccus" and A. polyphaga Linc-AP1 were kindly provided by T. J. Rowbotham. Three days' coculture of bacterial strains with A. polyphaga in PYG, corresponding to about 10 6 bacteria/ml, was used for inoculation of 5 ϫ 10 5 A. polyphaga organisms/ml. Cocultures in PAS were incubated at 32°C for 8 and 36 h, while PYG has been used for longer incubation (144 h), as amoebal lysis occurred after 3 to 5 days in PAS in the presence of Parachlamydia strains. Amoebae were washed twice in PAS (two centrifugation steps at 179 ϫ g for 10 min; He...

Recovery of an environmental chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.

“…Thus, the developmental cycle of Parachlamydia sp. UV-7 was highly similar to the developmental cycle of P. acanthamoebae, which has previously been studied in some detail (Greub & Raoult, 2002a). A crescent-shaped developmental stage, described by Greub & Raoult (2002a) was, however, rarely observed, even at 7 days post-infection (p.i.…”

Simkania negevensis, an insight into the biology and clinical importance of a novel member of the Chlamydiales order

“…Based on culture observations, S. negevensis does not seem to induce cell lysis, or at least not to the same extent as what is observed for other members of the Chlamydiales order, such as W. chondrophila (KebbiBeghdadi et al, 2011). In comparison, C. trachomatis particles are released between 30 and 68 h post-infection depending on the strain (Miyairi et al, 2006), W. chondrophila particles are released 48-72 h upon cell lysis (Goy et al, 2008) and P. acanthamoebae induces lysis of infected amebae after 72-96 h depending on the initial multiplicity of infection (MOI) and incubation temperature (Greub & Raoult, 2002b;Greub et al, 2003b). However, despite the apparent absence of cell lysis, some particles seem to be released in the extracellular environment, as suggested by the presence of S. negevensis particles in the supernatant of infected insect cell cultures capable of inducing a second round of infection (Sixt et al, 2012).…”

Proteomic aspects of Parachlamydia acanthamoebae infection in Acanthamoeba spp.

“…The latter is the infectious stage, which accumulates in the vacuole until the host cell is lysed, thereby liberating the elementary bodies, which can then start another round of infection. Alternatively, the bacteria can also be expelled from the cell in a vesicle (Greub and Raoult, 2002b). In contrast to C. pneumoniae and C. trachomatis, however, P. acanthamoebae has an astonishingly wide host range and cannot only infect Acanthamoeba but also distantly related or even unrelated free-living amoebae like Balamuthia mandrillaris, Hartmannella vermiformis and Naegleria spp.…”