Intracellular Behaviour of Three Legionella pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky

Abstract

Legionella pneumophila is a facultative intracellular pathogen found in aquatic environments as planktonic cells within biofilms and as intracellular parasites of free-living amoebae such as Acanthamoeba castellanii. This pathogen bypasses the elimination mechanism to replicate within amoebae; however, not all amoeba species support the growth of L. pneumophila. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to possess the ability to eliminate the L. pneumophila strain Paris. Here, we study the intracellular behaviour of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky and compare this strain to A. castellanii and W. magna Z503, which are used as controls. We observe the intracellular growth of strain Lens within W. magna Z503 and A. castellanii at 22 °C and 37 °C. Strain Paris grows within A. castellanii at any temperature, while it only grows at 22 °C within W. magna Z503. Strain Philadelphia proliferates only within A. castellanii at 37 °C. Within W. magna C2c Maky, none of the three legionella strains exhibit intracellular growth. Additionally, the ability of W. magna C2c Maky to decrease the number of internalized L. pneumophila is confirmed. These results support the idea that W. magna C2c Maky possesses unique behaviour in regard to L. pneumophila strains.

Keywords: Legionella; biological biocide; cooling towers; free-living amoebae.

Figure 1

L. pneumophila survival in coculture medium at 22 °C (a) and 37 °C (b). Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean).

Figure 2

Amoeba survival at 22 °C (a) and 37 °C (b) in coculture medium in the presence or absence of the three L. pneumophila strains (Lens, Paris, and Philadelphia). The red bar is the detection limit of the Malassez cell counting. Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean).

Figure 3

Comparison of the evolution of the number of intracellular L. pneumophila cells (Lens, Paris, and Philadelphia) per amoeba cell (A. castellanii, W. magna C2c Maky, and W. magna Z503). Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean). (a) L. pneumophila number per A. castellanii cell at 22 °C (n = 9 for Lp Lens and Paris, n = 15 for Lp Philadelphia); (b) L. pneumophila number per A. castellanii cell at 37 °C (n = 9); (c) L. pneumophila number per W. magna cell (C2c and Z503) at 22 °C (n = 9 for Lp Lens and Paris, n = 15 for Lp Philadelphia); (d) L. pneumophila number per W. magna cell (C2c and Z503) at 37 °C (n = 9).

Figure 4

Comparison of the evolution of the number of L. pneumophila cells in the presence or absence of amoeba cells (alone, or in presence of A. castellanii, W. magna C2c Maky, or W. magna Z503). Results are expressed as the mean +/− 95% CI (Confidence Interval based on the standard error of the mean). (a) L. pneumophila Lens at 22 °C (n = 9); (b) L. pneumophila Lens at 37 °C (n = 9); (c) L. pneumophila Paris at 22 °C (n = 9); (d) L. pneumophila Paris at 37 °C (n = 9); (e) L. pneumophila Philadelphia at 22 °C (n = 15); (f) L. pneumophila Philadelphia at 37 °C (n = 9).

Figure 5

Optical microscopy observation using Gimenez staining of A. castellanii (A–C), W. magna C2c Maky (D–F), and W. magna Z503 (G–I) infected with L. pneumophila Philadelphia at 37 °C. Photos of the co-cultures were acquired at T₀ (A,D,G), T₀ + 48 h (B,E,H), and T₀ + 96 h (C,F,I).