Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes

Abstract

Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449-2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.

FIG. 1

Multiplication of different Legionella species in MM6 and influence of coculture with A. castellanii. Data are means ± 95% confidence limits from three experiments. ∗, P < 0.05; ∗∗, P < 0.01; n.s., not significant.

FIG. 2

Multiplication of L. dumoffii in MM6 in coculture with uninfected and L. dumoffii-infected A. castellanii (A. cast.). Data are means ± 95% confidence limits from three experiments. ∗∗, P < 0.01.

FIG. 3

Multiplication of L. dumoffii in MM6 coincubated with supernatant obtained from a coculture of L. dumoffii- or L. steigerwaltii-infected MM6 with uninfected A. castellanii. Control, coculture supernatant from uninfected MM6 and A. castellanii. Data are means ± 95% confidence limits from three experiments. ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 4

Multiplication of L. longbeachae in MM6 coincubated with supernatant obtained from a coculture of L. dumoffii-, L. longbeachae-, or L. steigerwaltii-infected MM6 with uninfected A. castellanii. Data are means ± 95% confidence limits from three experiments. ∗, P < 0.05.

FIG. 5

Multiplication of L. steigerwaltii in MM6 coincubated with supernatant obtained from a coculture of L. dumoffii-, L. longbeachae-, or L. steigerwaltii-infected MM6 with uninfected A. castellanii. Data are means ± 95% confidence limits from three experiments.

FIG. 6

Influence of NaCl content of coculture medium on intracellular replication of L. dumoffii in MM6. Data are means ± 95% confidence limits from three experiments.