Abstract
Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.
Highlights
Animal models of infection have proved invaluable in the determination of bacterial virulence factors
It is shown that a number of aspects of macrophage infection can be recapitulated in the G. mellonella model, including the role of the Dot/Icm T4BSS in virulence and bacterial replication and the localization of the Dot/Icm-effector SidC
We demonstrate that a chemical inhibitor of actin polymerization significantly reduces larval mortality, mimicking results obtained in macophages[47] and supporting evidence that internalization of the bacteria is required to cause larval mortality
Summary
Animal models of infection have proved invaluable in the determination of bacterial virulence factors. Mammalian hosts have been characterized as models for human Legionella infection including mice and guinea pigs[24,25]. Several invertebrate models have been developed for Legionella pneumophila infection including Caenorhabditis elegans[28], Drosophila melanogaster[29] and several species of amoebae[30,31,32]. These models have weaknesses, virulence in the C. elegans system is not Dot/Icm-dependent[28], limiting the utility of this model. Note: Incubation of plates for 4 days significantly increases the virulence of L. pneumophila in G. mellonella over incubation for 3 days.
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