Legionella pneumophila is a gram-negative bacteria found ubiquitously in fresh water environment. When inhaled by humans, Legionella enters alveolar macrophages and establishes a replicative niche. Infections can eventually cause a severe form of pneumonia.
The type IV secretion system (T4SS) is one of the secretion systems used by many important bacterial pathogens to translocate effector proteins that interact with host factors to subvert host cellular processes. T4SS is categorized into two subgroups, type IVA (T4ASS) and type IVB (T4BSS) secretion systems according to evolutionary related bacterial conjugation systems. Legionella has a Dot/Icm T4BSS which is essential for a number of virulence traits including replication within host cells.
Key players: Bacterial effector proteins
Legionella delivers a large array of effector proteins into host cells via the Dot/Icm T4BSS. The number of identified effector proteins drastically expanded in the past years (almost 1/10 of the whole genome!). This indicates that this pathogen may possesses the complexity of regulation systems in host cells. The effectors can build functional networks and can be finely tuned over time according the stages of infection.
To understand the elegance of bacterial strategies to manipulate host cells, we focus on the function of effector proteins. Starting from systematic search for Legionella effectors, we now focus on the molecular roles of a subset of effector proteins. By using genetic, biochemical, and cell biology techniques, we are examining their role in the infected cells and how their function is regulated.
We recently found a Legionella effector protein LubX which functions as an E3 ubiquitin ligase in the cells (Kubori et al., Mol. Microbiol. 2008). Interestingly LubX works as a negative regulator of another Legionella effector protein SidH by subjecting this protein to ubiquitin-proteasome mediated degradation (Kubori et al., PLoS Pathogens 2010). Over the past years, modulation of host ubiquitin pathways have been found to be a crucial and broadly obtained bacterial strategy to hijack host cell functions in many pathogenic bacteria.
The regulation found for SidH has conceptual importance for its “destructive approach” which is likely a common strategy for bacteria to destroy their own proteins in infected cells that utilize host cellular function for their benefit. This work also gives novel insight into effector studies as LubX is the first described “metaeffector” an effector protein that regulates another bacterial effector protein. This means that targets of effector proteins are not always host factors. Legionella is a good model organism to studyeffector networks.
We anticipate that numerous processes facilitate temporal and spatial regulation of Legionella effectors. By examining when, where and how the effectors work inside infected cells, further details of the molecular mechanisms used for bacterial infection will be clarified.
How do bacteria translocate their own proteins directly into the host cell cytosol? To understand this at a molecular level, we have been working on structural analyses of the Legionella Dot/Icm secretion system. The X-ray crystal structure of the one of the core component DotD showed a striking resemblance to the structure of a subdomain of secretins which form outer membrane channels in the type II and type III secretion systems (Nakano et al., PLoS Pathogens 2010). This finding, together with other lines of evidence, highlights the mosaic nature of the T4BSS in an evolutionary aspect.
We also focus on the structure and assembly mechanism of the entire/sub complexes of the Dot/Icm secretion system. We are interested in their function and regulation mechanisms as a transporter. Using genetics, biochemistry, electron microscopy, and X-ray crystallography, we aim to understand the structure and function of the Legionella T4BSS.
Whereas Legionella-related bacteria (Legionellae) all carry well-conserved T4BSS, effector proteins are usually not related at the amino acid sequence level to proteins found in other organisms. The question is where did these effectors come from? One place worth examining is the genome of Acanthamoeba, which is a natural protozoan host of Legionella, and those of bacteria living in Acanthamoeba. Acanthamoeba isolated from the environment often carry bacteria inside. Acanthamoeba may act as a melting pot for organisms living inside, including Acanthamoeba giant viruses, mutualistic endosymbiotic bacteria and Legionella. Bearing this idea in mind, we are analyzing interaction between free-living amoeba and intracellular bacteria at molecular level.