The roles of the Legionella pneumophila effectors PieE and LtpG in formation and maintenance of the Legionella containing vacuole

Legionella pneumophila is the major causative agent of Legionnaires' disease, which is a severe, potentially fatal, form of pneumonia. The bacteria are transmitted via inhalation of contaminated water droplets (e.g. from air-conditioning units or swimming pools). The elderly and immunocompromised are most susceptible to infection, making Legionella a major health risk in hospitals and communal facilities (e.g. nursing homes). The potentially fast and wide dissemination of Legionella by airflow can lead to large outbreaks, as was seen in Edinburgh and Stoke-on-Trent in 2012, affecting dozens to hundreds of persons. Infectious sources of these outbreaks are often hard to trace and therefore difficult to eradicate, making Legionella an important health risk for densely-populated areas.

Following its inhalation, Legionella enters lung cells where it establishes a unique intracellular niche that protects the bacterium from destruction, allowing its own survival and multiplication. The ability of the bacterium to persist within human cells is mediated by special bacteria proteins, known as effectors, which are injected from the bacterial cell into the human cell where they interact with host cell proteins and re-programme cellular processes for the benefit of the bacterium. Understanding the mechanism by which Legionella effectors subvert host cell functions is essential for development of effective prevention and treatment strategies. By focusing our investigation on two important effectors, PieE and LtpG, we aim to reveal fundamental mechanisms employed by the bacteria to thrive in their host cells and cause disease.

The identification of host cell interaction partners of effectors is essential to understand their function. We developed a novel method that allows discovery of effector signalling complexes under physiological infection conditions. Our method relies on a cell line stably expressing the biotin ligase BirA from E. coli. Following infection with L. pneumophila expressing an effector fused to a Bio-tag consisting of a His6 tag and a BirA specific biotinylation site, the effector-host protein complexes are stabilized by chemical cross-linking and purified under denaturing conditions in two steps using Nickel and Streptavidin columns (Tandem affinity, TA, chromatography). Proteins in the complexes are then identified by mass spectrometry (MS). Unspecific hits are eliminated by cross-comparison with results obtained from infections with L. pneumophila containing the empty pBio plasmid and an unrelated Bio-tagged effector. Specific interactions are confirmed by two hybrid systems, co-IP, pull downs and ELISA. In addition we will:

1. Use live cell imaging to track interaction partners, vesicular trafficking and, employing a new split-GFP system, the effectors during infection.
2. Express host proteins and effectors fused to the miniSOG-tag, a bi-functional tag for correlated, high resolution IF and TEM.
3. Use TR-FRET to characterise protein-protein interactions within the effector complexes.
4. Use chemical probes and click chemistry to determine the effect of effector binding on the post-translational modification of Rabs.
5. Use RNAi to deplete host cell proteins and site directed mutagenesis to delete effector genes from Legionella (and complementation) to study their roles during infection.
6. Purify recombinant proteins for biochemical and cell-free vesicle tethering assays and structural studies.
7. Study the coatomer complex, p62, autophagy, MAP kinase and NF-kB dependent survival signalling and regulation of cytokine expression using IF, WB and ELISA.

Legionella pneumophila is the etiological agent of the potentially fatal Legionnaires' disease. Outbreaks are often linked to human-made aquatic environments (e.g. air-conditioning water systems and swimming pools) where L. pneumophila persists in amoeba and biofilms. The elderly and immunocompromised are most susceptible to infection, making Legionella a major health risk in hospitals and communal facilities (e.g. nursing homes). The global demographic change towards an older population most probably accounts for the increased incidence of Legionella infections during the last 30 years. The potentially fast and wide dissemination of Legionella by airflow can lead to large outbreaks, as was seen in Edinburgh and Stoke-on-Trent in 2012, affecting dozens to hundreds of persons. Infectious sources of these outbreaks are often hard to trace and therefore difficult to eradicate, making Legionella an important health risk for densely-populated areas.
Key to Legionella infection is the formation of a replicative vacuole, called the Legionella-containing vacuole (LCV), in amoeba and macrophages. The formation of the LCV is mediated by the translocation of hundreds of type IV secretion system (T4SS) effectors and subversion of endosomal trafficking. Importantly, the formation and maintenance of the LCV and the ability of the pathogen to prevent its fusion with lysosomes and autophagosomes is an infection strategy shared by several other important human pathogens including Salmonella, Rickettsia and Chlamydia.

In the short term this research is unlikely to impact on policies or the wider public. However, in the longer term we aim to provide important insights into the mechanism by which Legionella subverts its host cells and establishes and maintains the LCV. As this is key to Legionella infection, we believe the data could impact on development of new measures to control persistence of Legionella in the environment or of therapies to treat disease. This will directly impact on policy makers and enhance the public health and quality of life.