Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Abstract

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

FIG. 1.

Native preparations of B. mandrillaris cultured without or with L. pneumophila strain 130b for 48 h, 72 h, and 96 h. Primary magnification, ×164.

FIG. 2.

Transmission electron micrographs of B. mandrillaris cultured for 24 h without (a) or with (b to d) L. pneumophila 130b. Small arrows designate rough endoplasmic reticulum adjoined to the L. pneumophila-containing vacuole; the large arrow designates dividing bacteria.

FIG. 3.

Transmission electron micrographs of B. mandrillaris cultured for 72 h without (a) or with (b to d) L. pneumophila 130b. Small arrows designate rough endoplasmic reticulum adjoined to the L. pneumophila-containing vacuole; the large arrow designates dividing bacteria.

FIG. 4.

Intracellular infection by different L. pneumophila strains. (Top) CFU of L. pneumophila 130b (L.p.) in cultures of bacteria alone or in coculture with B. mandrillaris amebas. By 72 h, bacterial numbers in cultures without amebas had dropped below the detection level. (Bottom) The L. pneumophila JR32 wild type and L. pneumophila JR32 dotA mutant in coculture with B. mandrillaris amebas. Results are the means of CFU ± standard deviations for triplicate samples and are representative of results of three independent experiments.