Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease

Abstract

The effect of inhaled amoebae on the pathogenesis of Legionnaires' disease was investigated in vivo. A/J mice, which are susceptible to replicative Legionella pneumophila infections, were inoculated intratracheally with L. pneumophila (10 6 bacteria per mouse) or were coinoculated with L. pneumophila (10 6 bacteria per mouse) and Hartmannella vermiformis (10 6 amoebae per mouse). The effect of coinoculation with H. vermiformis on bacterial clearance, histopathology, cellular recruitment into the lung, and intrapulmonary levels of cytokines including gamma interferon and tumor necrosis factor alpha was subsequently assessed. Coinoculation with H. vermiformis significantly enhanced intrapulmonary growth of L. pneumophila in A/J mice. Histopathologic and flow cytometric analysis of lung tissue demonstrated that while A/J mice inoculated with L. pneumophila alone develop multifocal pneumonitis which resolves with minimal mortality, mice coinoculated with H. vermiformis develop diffuse pneumonitis which is associated with diminished intrapulmonary recruitment of lymphocytes and mononuclear phagocytic cells and significant mortality. Furthermore, coinoculation of mice with H. vermiformis resulted in a fourfold enhancement in intrapulmonary levels of gamma interferon and tumor necrosis factor alpha compared with mice infected with L. pneumophila alone. The effect of H. vermiformis on intrapulmonary growth of L. pneumophila in a resistant host (i.e., BALB/c mice) was subsequently evaluated. While BALB/c mice do not develop replicative L. pneumophila infections following inoculation with L. pneumophila alone, there was an eightfold increase in intrapulmonary L. pneumophila in BALB/c mice coinoculated with H. vermiformis. These studies, demonstrating that intrapulmonary amoebae potentiate replicative L. pneumophila lung infection in both a susceptible and a resistant host, have significant implications with regard to the potential role of protozoa in the pathogenesis of pulmonary diseases due to inhaled pathogens and in the design of strategies to prevent and/or control legionellosis.

Effects of co‐culture of amoebae with indoor microbes on their cytotoxic and proinflammatory potential

“…Thus, the effect of cocultivation with amoebae with respect to TNF-production was again synergistic. This same phenomenon has been observed earlier for the gram negative bacterium Legionella pneumophila, where the exposure to amoeba-grown bacteria induced 10 times higher concentrations of TNF-in the bronchoalveolar lavage suspension of live mice than either the bacterium or the amoebae on their own (Brieland et al, 1996).…”

“…The results of exposure to amoebal lysate depended on the pathogenicity of the amoeba (Shin et al, 2000(Shin et al, , 2001. The pathogenic clinical strains of amoebae have been shown to be bioactive, resulting in elevated levels of cytotoxicity and cytokine production (Brieland et al, 1996(Brieland et al, , 1997Marciano-Cabral and Toney, 1998;Shin et al, 2000Shin et al, , 2001. However, in these studies the target cells have usually been exposed to live amoebae.…”

Heterogeneity in chlorine susceptibility forLegionella pneumophilareleased fromAcanthamoebaandHartmannella

“…pneumophila with H. vermiformis significantly enhances intrapulmonary growth of Leg. pneumophila in mice (Brieland et al 1996), whereas co-cultures of Leg. pneumophila and Acanthamoeba royreba do not (Tyndall and Domingue 1982).…”

The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease

“…A/J mice were inoculated intratracheally with L. pneumophila-infected H. vermiformis (10 6 amoebae containing 10 5 bacteria per mouse harvested after 6 h of coculture) or were inoculated with L. pneumophila (10 5 bacteria per mouse) and/or uninfected H. vermiformis (10 6 amoebae per mouse). At 24 h postinoculation, the mice were sacrificed and the lungs were lavaged with phosphate-buffered saline (1.6 ml per mouse) as previously described (9). IFN-␥ and TNF-␣ were assessed in bronchoalveolar lavage fluid by enzyme-linked immunosorbent assay (ELISA) and bioassay, respectively (7)(8)(9).…”

“…At 24 h postinoculation, the mice were sacrificed and the lungs were lavaged with phosphate-buffered saline (1.6 ml per mouse) as previously described (9). IFN-␥ and TNF-␣ were assessed in bronchoalveolar lavage fluid by enzyme-linked immunosorbent assay (ELISA) and bioassay, respectively (7)(8)(9). As is shown in Table 1, intrapulmonary levels of IFN-␥ and TNF-␣ were significantly enhanced in A/J mice inoculated with L. pneumophila-infected H. vermiformis compared with levels in mice inoculated with the bacteria alone.…”