Impact of inter-amoebic phagocytosis on the L. pneumophila growth.

Free-living amoebae are known to act as replication niches for the pathogenic bacterium Legionella pneumophila in freshwater environments. However, we previously reported that some strains of the Willaertia magna species are more resistant to L. pneumophila infection and differ in their ability to support its growth. From this observation, we hypothesize that L. pneumophila growth in environment could be partly dependent on the composition of amoebic populations and on the possible interactions between different amoebic species. We tested this hypothesis by studying the growth of L. pneumophila and of a permissive free-living amoeba, Vermamoeba vermiformis (formerly named Hartmannella vermiformis), in co-culture with or without other free-living amoebae (Acanthamoeba castellanii and W. magna). We demonstrate the occurrence of inter-amoebic phagocytosis with A. castellanii and W. magna being able to ingest V. vermiformis infected or not infected with L. pneumophila. We also found that L. pneumophila growth is strongly impacted by the permissiveness of each interactive amoeba demonstrating that L. pneumophila proliferation and spread are controlled, at least in part, by inter-amoebic interactions.

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella.

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.

From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella.

The 1976 outbreak of Legionnaires' disease led to the discovery of the intracellular bacterial pathogen Legionella pneumophila. Given their impact on human health, Legionella species and the mechanisms responsible for their replication within host cells are often studied in alveolar macrophages, the primary human cell type associated with disease. Despite the potential severity of individual cases of disease, Legionella are not spread from person-to-person. Thus, from the pathogen's perspective, interactions with human cells are accidents of time and space-evolutionary dead ends with no impact on Legionella's long-term survival or pathogenic trajectory. To understand Legionella as a pathogen is to understand its interaction with its natural hosts: the polyphyletic protozoa, a group of unicellular eukaryotes with a staggering amount of evolutionary diversity. While much remains to be understood about these enigmatic hosts, we summarize the current state of knowledge concerning Legionella's natural host range, the diversity of Legionella-protozoa interactions, the factors influencing these interactions, the importance of avoiding the generalization of protozoan-bacterial interactions based on a limited number of model hosts and the central role of protozoa to the biology, evolution, and persistence of Legionella in the environment.

Amoebae and Legionella pneumophila in saline environments.

Amoeboid protists that harbor bacterial pathogens are of significant interest as potential reservoirs of disease-causing organisms in the environment, but little is known about them in marine and other saline environments. We enriched amoeba cultures from sediments from four sites in the New England estuarine system of Mt. Hope Bay, Massachusetts and from sediments from six sites in the Great Salt Lake, Utah. Cultures of amoebae were enriched using both minimal- and non-nutrient agar plates, made with fresh water, brackish water or saltwater. Recovered amoeba cultures were assayed for the presence of Legionella species using nested polymerase chain reactions (PCR) and primers specific for the genus. Positive samples were then screened with nested amplification using primers specific for the macrophage infectivity potentiator surface protein (mip) gene from L. pneumophila. Forty-eight percent (185 out of 388) of isolated amoeba cultures were positive for the presence of Legionella species. Legionella pneumophila was detected by PCR in 4% of the amoeba cultures (17 out of 388), and most of these amoebae were growing on marine media. Our results show that amoebae capable of growing in saline environments may harbor not only a diverse collection of Legionella species, but also species potentially pathogenic to humans.

A faster and more accurate assay for intracellular replication of Legionella pneumophila in amoebae hosts.

A faster, more sensitive, and more accurate method to study intracellular replication of Legionella pneumophila in amoeboid hosts is described. This assay relies on an automated plate-reader to examine the intracellular growth and release of bacteria in real-time. Our experiments using this method have already revealed new insights into the kinetics of intracellular replication of L. pneumophila in Acanthamoeba castellanii.

Type IV pili and type II secretion play a limited role in Legionella pneumophila biofilm colonization and retention.

Legionellae colonize biofilms in building water systems, yet little is known about their interaction with the organisms in these microbial communities. The role of Legionella pneumophila type IV pili and the type II secretion pre-pilin peptidase was evaluated in a model biofilm system. L. pneumophila strains 130b (wild-type), BS100 (a type IV pili mutant) and NU243 (a pre-pilin peptidase mutant) were assessed for attachment and retention in an established biofilm. Strains 130b and NU243 colonized the biofilm at a similar level while BS100 attached at a tenfold lower level. Over time, NU243 dropped below the level of detection while BS100 remained in the biofilm throughout the course of the experiment. The wild-type strain decreased but remained at a considerably higher level than either of the mutants. Inclusion of amoebae with BS100 allowed for attachment and retention at a level similar to 130b. NU243, which displays reduced intracellular replication, was able to establish itself and persist in the presence of amoebae. Thus, type IV pili and the pre-pilin peptidase facilitate L. pneumophila colonization of biofilms but are not required in the presence of a host for intracellular replication.

[Intra-amoebal development of Legionella pneumophila and the potential role of amoebae in the transmission of Legionnaires' disease]. / Multiplication intra-amibienne de Legionella pneumophila et rôle potentiel des amibes dans la transmission de la légionellose.

Legionnaires' disease is one of the major infectious risks related to hospital water systems. It is commonly accepted, that the disease is transmitted to man mostly by inhalation of water aerosols contaminated by Legionella pneumophila. The ability of L. pneumophila to multiply intracellularly within some amoebae better explains the ecology, the pathogenicity, and the virulence of this bacterium against human alveolar macrophages. The presence of these amoebae in water systems located where cases of Legionnaire's disease broke out, partly explains the difficulty in eradicating Legionella. Some studies also show that amoebae can play a major role in the transmission of the disease to man. Some other studies point out that inhaled amoebae could be involved in the pathogenesis of Legionnaire's disease. Future strategies to prevent the transmission of Legionella will probably have to include efficient treatments against amoebae.

Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells.

In contrast to Legionella pneumophila, little is known about the pathogenesis of other legionellae species that are capable of causing Legionnaires' disease. In this report, we contrast L. pneumophila and L. micdadei for their cytopathogenicity and intracellular replication within mammalian and protozoan cells. We show by transmission electron microscopy that L. micdadei replicates within an endoplasmic reticulum (RER)-free phagosome within human macrophages, alveolar epithelial cells, and within the protozoan Hartmannella vermiformis. In contrast, L. pneumophila replicates within a RER-surrounded phagosome within the same host cells. In contrast to replication of L. pneumophila within Acanthamoebae polyphaga, L. micdadei does not replicate within this protozoan host. Despite the prolific intracellular replication, L. micdadei is less cytopathogenic to all host cells than L. pneumophila. Since both species replicate intracellularly to a similar level, we have examined whether the reduced cytopathogenicity of L. micdadei is due to a reduced capacity to induce apoptosis or pore formation-mediated necrosis, both of which contribute to killing of the host cell by L. pneumophila. The data show that both species induced apoptosis-mediated killing of mammalian cells to a similar level. In contrast to L. pneumophila, expression of the pore-forming toxin by L. micdadei and its necrotic effect on macrophages and alveolar epithelial cells is undetectable. This has been further confirmed showing that L. micdadei is completely defective in contact-dependent haemolysis of RBCs, an activity mediated by the pore-forming toxin. Finally, in contrast to L. pneumophila, there was no significant intrapulmonary replication of L. micdadei in the A/J mice animal model. Our data show dramatic differences between L. pneumophila and L. micdadei in intracellular replication, cytopathogenicity, and infectivity to mammalian and protozoan cells.