Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells

Abstract

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophila in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phases of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 ,ug/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D-and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Legionella pneumophila: an aquatic microbe goes astray

“…In contrast to previously published results obtained with Acanthamoeba polyphaga [33] data reported recently suggest the requirement for host protein synthesis during bacterial uptake by A. castellanii [34]. This reduction in uptake was enhanced by simultaneous addition of cytochalasin D. These ¢ndings con¢rm the proposed heterogeneity of uptake mechanisms by di¡erent protozoan hosts [27,35,36]. Apparently A. cas-tellanii uses multiple mechanisms for bacterial uptake, while H. vermiformis may use only receptor-mediated pinocytosis.…”

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

“…Although it has been suggested that entry of L. pneumophila into amoebae may not require actin filaments (King et al ., 1991;Moffat and Tompkins, 1992), we found that pathogen uptake is an actin-mediated process in Dictyostelium . Treatment of amoebae with a concentration of latrunculin-A sufficient to induce global depolymerization of actin filaments (Gerisch et al ., 2004) reversibly blocked detectable entry of bacteria into the cells.…”

“…Many approaches have demonstrated that actin filaments play an essential role in the uptake of L. pneumophila by mammalian cells. An early study showed that cytochalasin D, an inhibitor of actin filament formation, blocked L. pneumophila replication in the monocyte-like cell line U937 (King et al, 1991). However, there has been uncertainty about the role of actin in the uptake of L. pneumophila by protozoa and amoebae, because higher concentrations of cytochalasin D than those inhibitory for monocytes failed to block growth of L. pneumophila in H. veriformis (King et al, 1991) or A. castellani (Moffat and Tompkins, 1992;Kohler et al, 2000).…”

“…An early study showed that cytochalasin D, an inhibitor of actin filament formation, blocked L. pneumophila replication in the monocyte-like cell line U937 (King et al, 1991). However, there has been uncertainty about the role of actin in the uptake of L. pneumophila by protozoa and amoebae, because higher concentrations of cytochalasin D than those inhibitory for monocytes failed to block growth of L. pneumophila in H. veriformis (King et al, 1991) or A. castellani (Moffat and Tompkins, 1992;Kohler et al, 2000). Indeed, it has been suggested that depolymerization of the actin cytoskeleton may be part of the mechanism that allows L. pneumophila to invade H. veriformis (Venkataraman and Kwaik 2000).…”

Multiplication of Legionella pneumophila in HeLa Cells in the Presence of Cytoskeleton and Metabolic Inhibitors

“…Yamamoto et al (36) and Husmann and Johnson (18) used cytochalasin D in order to block the phagocytosis of L. pneumophila by murine and guinea pig macrophages, respectively. It has also been shown that cytochalasin D inhibited intracellular multiplication of L. pneumophila in the U937 monocyte model, but had no effect on the intracellular multiplication of legionellas in the axenic amoeba Hartmannella vermiformis (20). Moreover the same authors described the inhibition of L. pneumophila multiplication in both U937 monocytes and H. vermiformis by methylamine, a metabolic inhibitor which affects pinocytosis acting on transglutaminase.…”