Identification of a Novel Adhesion Molecule Involved in the Virulence ofLegionella pneumophila

Chang, Bin; Kura, Fumiaki; Amemura-Maekawa, Junko; Koizumi, Nobuo; Watanabe, Haruo

doi:10.1128/iai.73.7.4272-4280.2005

Cited by 42 publications

(41 citation statements)

References 57 publications

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“…The ease of G. mellonella infection allows for rapid, medium throughput screens that could be used to compare the virulence of various Legionella species and strains and could be used to further analyze previously identified virulence factors such as adhesion molecules 58 or the type 2 secretion system 59 which are required for virulence in other models. In addition, use of this model will allow the identification and further characterization of novel virulence factors including secreted and translocated effector proteins.…”

Section: Discussionmentioning

confidence: 99%

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Harding

Schroeder

Collins

et al. 2013

JoVE

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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.

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Section: Discussionmentioning

confidence: 99%

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Harding

Schroeder

Collins

et al. 2013

JoVE

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“…In addition to infecting macrophages, L. pneumophila is able to invade and grow within alveolar epithelial cells (25,43,64,80). Therefore, we next determined the relative ability of an lbtA mutant to infect A549 cells, an alveolar epithelial line that is known to support L. pneumophila infection (18,36,43,69,70,103). However, the mutant infected these cells as well as parental 130b did (not shown).…”

Section: Purification Of Legiobactin and Detection Of Ferrilegiobactinmentioning

confidence: 99%

Purification of Legiobactin and Importance of This Siderophore in Lung Infection byLegionella pneumophila

Allard

Dao

Sanjeevaiah³

et al. 2009

Infect Immun

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When cultured in a low-iron medium, Legionella pneumophila secretes a siderophore (legiobactin) that is both reactive in the chrome azurol S (CAS) assay and capable of stimulating the growth of iron-starved legionellae. Using anion-exchange high-pressure liquid chromatography (HPLC), we purified legiobactin from culture supernatants of a virulent strain of L. pneumophila. In the process, we detected the ferrated form of legiobactin as well as other CAS-reactive substances. Purified legiobactin had a yellow-gold color and absorbed primarily from 220 nm and below. In accordance, nuclear magnetic resonance spectroscopy revealed that legiobactin lacks aromatic carbons, and among the 13 aliphatics present, there were 3 carbonyls. When examined by HPLC, supernatants from L. pneumophila mutants inactivated for lbtA and lbtB completely lacked legiobactin, indicating that the LbtA and LbtB proteins are absolutely required for siderophore activity. Independently derived lbtA mutants, but not a complemented derivative, displayed a reduced ability to infect the lungs of A/J mice after intratracheal inoculation, indicating that legiobactin is required for optimal intrapulmonary survival by L. pneumophila. This defect, however, was not evident when the lbtA mutant and its parental strain were coinoculated into the lung, indicating that legiobactin secreted by the wild type can promote growth of the mutant in trans. Legiobactin mutants grew normally in murine lung macrophages and alveolar epithelial cells, suggesting that legiobactin promotes something other than intracellular infection of resident lung cells. Overall, these data represent the first documentation of a role for siderophore expression in the virulence of L. pneumophila.

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“…However, the overlap between the Ags identified with these two methods was very small, as only one Ag (Lpg0533, dihydrolipoamide succinyltransferase) was present in both sets of proteins. Likewise, none of the three L. pneumophila B cell Ags that were previously described using an approach similar to the l phage expression screen (Lpg2175, Lpg2327, and Lpg2803) (33) were among the Ags we identified in this study. It is possible that most of the Ags discovered by the l phage technique are expressed at only low levels in L. pneumophila and, therefore, were below the detection limit of the two-dimensional PAGE Western blot.…”

Section: Discussionmentioning

confidence: 69%

“…Moreover, some L. pneumophila genes are likely not represented in the l phage library, possibly because they were cleaved when the phage inserts were prepared. Perhaps the use of different restriction enzymes to generate the phage libraries accounts for the poor representation in our screen of previously identified Ags (33). Nevertheless, we have identified a total of 30 novel L. pneumophila B cell Ags.…”

Section: Discussionmentioning

confidence: 97%

“…Moreover, several of these Ags, identified using L. pneumophila-specific rabbit antisera, were also recognized by Abs found in sera of patients, indicating that L. pneumophila B cell Ags are, at least to a certain extent, conserved across host species (32). However, specific screens have resulted in the identification of only three distinct L. pneumophila protein B cell Ags to date, namely, the surface protein LaiA (Lpg2157), a T4SS substrate (Lpg2327), and the hypothetical protein Lpg2803 (33,34). However, the protective character of the Ab response directed against these proteins has not been evaluated.…”

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confidence: 99%

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Identification of Protective B Cell Antigens of Legionella pneumophila

Weber

Joller

Küntzel

et al. 2012

The Journal of Immunology

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Abs confer protection from secondary infection with Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires’ disease. In this study, we demonstrate that Ab-mediated protection is effective across L. pneumophila serogroups, suggesting that Abs specific for conserved protein Ags are sufficient to mediate this protective effect. We used two independent methods to identify immunogenic L. pneumophila protein Ags, namely, the screening of a λ phage library representing the complete L. pneumophila genome and two-dimensional gel electrophoresis combined with Western blot analysis and protein spot identification by mass spectrometry. A total of 30 novel L. pneumophila B cell Ags were identified, the majority of which are located in or associated with the bacterial membrane, where they are accessible for Abs and, therefore, likely to be relevant for Ab-mediated protection against L. pneumophila. Selected B cell Ags were recombinantly expressed and tested in a vaccination protocol. Mice immunized with either single-protein Ags or an Ag combination showed reduced bacterial titers in bronchoalveolar lavage and lung after L. pneumophila challenge. To determine the clinical relevance of these findings, we tested Legionnaires’ disease patient sera for reactivity with the identified L. pneumophila Ags. The recognized Ags were indeed conserved across host species, because Abs specific for all three selected Ags could be detected in patient sera, rendering the identified protein Ags potential vaccine candidates.

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Identification of a Novel Adhesion Molecule Involved in the Virulence ofLegionella pneumophila

Cited by 42 publications

References 57 publications

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

Use of <em>Galleria mellonella</em> as a Model Organism to Study <em>Legionella pneumophila</em> Infection

Purification of Legiobactin and Importance of This Siderophore in Lung Infection byLegionella pneumophila

Identification of Protective B Cell Antigens of Legionella pneumophila

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