The genetic basis of Legionella pneumophila virulence

© 2016 Dr. Engku Nuraishah Huda E.ZainudinLegionella pneumophila utilizes a range of bacterial translocated effector proteins to manipulate complex host cell processes that aid intracellular replication and evasion of killing by their natural hosts, aquatic amoebae. These mechanisms also allow L. pneumophila to evade killing by human alveolar macrophages. Collectively, the effectors are essential in the development of an intracellular vacuole that supports bacterial replication and their secretion and translocation are strictly dependent on the Dot/Icm type IV secretion system. To understand the genetic basis of L. pneumophila intracellular replication in eukaryotic cells, we constructed and screened a library of 10,006 L. pneumophila IS903 transposon mutants for attenuated intracellular replication within the amoebae Acanthamoeba castellanii. From this large transposon screen, 34 mutants were identified, 21 of which carried insertions into the Dot/Icm Type IV secretion system. Insertions into seven novel genes were recovered, including lpw27511, vacB, potC and sdeC. Further investigation showed that the vacB and potC IS903 mutants were also significantly attenuated for replication in THP1 macrophages and that this phenotype could be complemented in trans by reintroducing native copies of vacB and potC on a plasmid. PotC encodes member of the ATP-Binding Cassette (ABC) Superfamily of transporter while VacB encodes member of RNR ribonuclease family. Additionally, we investigated the contribution of two Dot/Icm effector proteins, MavK and MavL to L. pneumophila pathogenesis. Confocal laser scanning microscopy (CLSM) showed that ectopically expressed GFP-MavK and GFP-MavL was distributed throughout the cytoplasm in HeLa cells and that expression led to disintegration of the Golgi apparatus. Epitope tagged 4HA-MavK and 4HA-MavL delivered by the Dot/Icm secretion system during L. pneumophila infection also showed a cytoplasmic distribution but with increased intensity at the cell periphery. To identify possible binding partners of MavK and MavL, we screened a HeLa cell cDNA library using the yeast two-hybrid system and MavK and MavL as bait. Nice-5, also known as UBE2Q1, a ubiquitin-conjugating enzyme E2Q family was unequivocally identified as a MavL binding partner, whereas MavK interacted with six possible host binding targets namely SCNM1(13hits), ATP50 (10 hits), CSPP1 (9 hits), ZNF263 (8 hits), MKRN1 (6 hits) and DTX3L (4 hits). The interaction between MavL and Nice-5 was confirmed by co-immunoprecipitation of epitope tagged MavL and native Nice-5. This latter work suggests that during L. pneumophila infection, MavL manipulates Nice-5 function, possible through a previously unrecognised ADP-ribose (ARTD8) binding domain. MavL is the latest effector suggested to influence host cell ubiquitination pathways in a manner that presumably aids L. pneumophila intracellular survival. Overall, this work has provided deeper insight into the genes that contribute to the ability of L. pneumophila to replicate in eukaryotic cells and cause disease