Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis survival within amoebal cysts

Abstract

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.

FIG. 1

Determination of percentage of acanthamoebae infected with S. negevensis by IPA. Infected amoebae stain dark blue with hyperimmune rabbit serum, peroxidase-conjugated swine anti-rabbit antibodies, and benzidine substrate. Stained and unstained amoebae are easily distinguished. Magnification, ×400.

FIG. 2

Transmission electron micrograph of a thin section of an A. polyphaga trophozoite infected with S. negevensis. Inclusion vesicles containing numerous or single RB and EB are seen. Bar = 1 μm. (Inset) Enlarged portion of the figure showing two inclusions containing somewhat elongated, condensed EB and rather pleomorphic RB. Bar = 0.3 μm.

FIG. 3

Growth curves of S. negevensis in A. polyphaga and of the amoebae themselves in the same cultures. (A) Infection of a relatively high-density amoebal culture at an MOI of 1, with S. negevensis purified from infected Vero cell cultures. (B) Results after dilution of the 15-day culture shown in panel A into fresh PYG medium (see text). Solid symbols, IFU of S. negevensis; open symbols, number of amoebae in the culture at the given time point. Bars indicate the standard deviation of the mean for amoebal counts and S. negevensis titers determined in triplicate. p.i., postinfection.

FIG. 4

Time course of S. negevensis nonproliferative infection (left panels) versus productive infection (right panels) of amoebae, as monitored by EM. (A, C, and E) One, 7, and 15 days postinfection, respectively. (B, D, and F) One, 10, and 16 days postinfection, respectively. Bar = 0.5 μm.

FIG. 5

Electron micrographs of A. polyphaga infected with S. negevensis, 2 days after encystation, demonstrating several possible interactions between the two organisms. (A) Precyst. (B to D) Double-walled cysts. Bars = 1 μm.