Citrobacter rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. Thus, C. rodentium has long been used as a model to understand the molecular basis of EPEC and EHEC infection in vivo. In this Review, we discuss recent studies in which C. rodentium has been used to study mucosal immunology, including the deregulation of intestinal inflammatory responses during bacteria-induced colitis and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. These insights should help to elucidate the roles of mucosal inflammatory responses and the microbiota in the virulence of enteric pathogens.

Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases this ribotype’s sensitivity to trehalose by >500 fold. Furthermore, dietary trehalose increases virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.

Clinicaltrials.gov: NCT01279265 and NCT01849991.

bLegionella pneumophila is a facultative intracellular human pathogen and the etiological agent of severe pneumonia known as Legionnaires' disease. Its virulence depends on protein secretion systems, in particular, the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication-permissive vacuole in macrophages. The analysis of the role of these systems and their substrates for pathogenesis requires easy-to-use models which approximate human infection. We examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for L. pneumophila infection. We found that the L. pneumophila strains 130b, Paris, and JR32 caused mortality of the G. mellonella larvae that was strain, infectious dose, growth phase, and T4SS dependent. Wild-type L. pneumophila persisted and replicated within the larvae, whereas T4SS mutants were rapidly cleared. L. pneumophila strain Lp02, which is attenuated in the absence of thymidine but has a functional T4SS, resisted clearance in G. mellonella up to 18 h postinfection without inducing mortality. Immunofluorescence and transmission electron microscopy revealed that L. pneumophila resided within insect hemocytes in a vacuole that ultrastructurally resembled the Legionella-containing vacuole (LCV) observed in macrophages. The vacuole was decorated with the T4SS effector and LCV marker SidC. Infection caused severe damage to the insect organs and triggered immune responses, including activation of the phenoloxidase cascade leading to melanization, nodule formation, and upregulation of antimicrobial peptides. Taken together, these results suggest that G. mellonella provides an effective model to investigate the interaction between L. pneumophila and the host.

Citation: Harding, C.R., Schroeder, G.N., Collins, J.W., Frankel, G. Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection. J. Vis. Exp. (81), e50964, doi:10.3791/50964 (2013). AbstractLegionella 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.