Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila

Abstract

Legionella pneumophila multiplied rapidly in guinea pig and rat alveolar macrophages but failed to grow when phagocytic activity was inhibited by pretreatment with 0.5 or 1.0 ,ug of cytochalasin D per ml. Attachment was not inhibited by cytochalasin D. No extracellular multiplication occurred when L. pneumophila were in close proximity to viable functional macrophages or even when the bacteria were attached to plasma membranes of the macrophages. Nonopsonized L. pneumophila were avidly phagocytized by alveolar macrophages. When bacteria were centrifuged onto a cell pellet, more than 85% of the phagocytes contained one or more bacteria within 15 min. In contrast, under the same conditions only approximately 15% of the macrophages contained nonopsonized Escherichia coi or Staphylococcus aureus. Phagocytosis of L. pneumophila by untreated guinea pig macrophages occurred by extension of pseudopodia around the bacteria in a classical manner. The failure of the bacteria to actively penetrate the phagocyte suggests that their intracellular survival must not depend on avoidance of a phagosome but rather on an inhibition of or resistance to subsequent microbicidal functions of the macrophage.Legionella pneumophila is a ubiquitous gram-negative bacterium which is an important cause of endemic and epidemic pneumonia (24,38). As a facultative intracellular parasite, the bacterium has a close relationship to macrophages in vivo.

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

“…An inhibiting effect on L. pneumophila internalisation in Acanthamoeba castellanii was reported by Moffat and Tompkins (23). Moreover, Elliot and Winn (7) and King et al (20) reported that the growth of L. pneumophila was greatly reduced in both guinea pig and rat macrophages and in human monocyte-like cells pretreated with cytochalasin D, suggesting that bacteria enter these cells by a type of microfilament-dependent phagocytosis. This does not appear to be the case for HeLa cells, where penetration does not seem to be a cytochalasin-sensitive endocytic process.…”

“…L. pneumophila entry and survival depend on the host cell physiological conditions and can consequently be influenced by several compounds that alter cell structures and metabolic functions. The importance of an intact functional cytoskeleton was demonstrated by Elliott and Winn (7) who observed that L. pneumophila could not grow in guinea pig and rat macrophage cultures in which phagocytic activity, but not bacterial attachment to the macrophage surface, was inhibited by cytochalasin D, an inhibitor of actin filament polymerisation. 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.…”

Autophagy is an immediate macrophage response to Legionella pneumophila

“…First, after a 5 min incubation with PE Legionella , only 21 ± 0.5% of the macrophages had ingested a bacterium, yet 54 ± 3% of the cells contained numerous Atg7-labelled donut-shaped compartments. Second, when actin polymerization was disrupted with cytochalasin D, phagocytosis of Legionella was decreased dramatically as expected (Elliott and Winn, 1986), yet 42 ± 1.7% of the macrophages still contained multiple Atg7decorated vacuoles within 5 min of infection, a frequency significantly greater than the 5.5 ± 1.5% of the uninfected control cells that did so ( Fig. 2B).…”

Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells

“…It seems that the entry of legionellae into amoebae generally is a very rapid process, that has been shown to be independent of microfilament polymerisation [15,32]. Conversely, phagocytosis of legionellae by macrophages can 305 be inhibited by treating the host cells with cytochalasins that prevent microfilament formation [35].…”