Characterization of The Viable but Non-Culturable Legionella pneumophila in Water and the Role of 3-Hydroxybutyrate Dehydrogenase in Its Formation
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Legionella pneumophila, the causative agent of Legionnaires’ disease (LD), is an intracellular pathogen of freshwater protozoa that can also persist in the environment as a free-living bacterium. L. pneumophila has many morphological forms that fit within a developmental cycle. In water, L. pneumophila enters into a viable but non-culturable (VBNC) state that is largely uncharacterized. VBNC cells were produced from two developmental L. pneumophila forms, stationary phase forms (SPFs) and mature infectious forms (MIFs) by suspension in double deionized (dd) or tap-water at 45°C. Electron microscopy results showed that VBNC cells have a unique morphology and that in tap water they lose their poly 3-hydroxybutyrate inclusion bodies. Both SPFs and MIFs lost culturability faster in dd- than in tap water, and addition of salts to dd-water prolonged L. pneumophila culturability and enhanced viability. However, MIFs retained higher viability in dd- and tap water (85% and 51%, respectively) than SPFs (5% and 20%, respectively) as determined by the BacLight vital stain. Only ~1 VBNC cell out of 105 of those produced from SPFs in tap water regained culturability via infection of Acanthamoeba. All VBNC cells, except for those produced from SPFs in dd-water, resisted both digestion inside Tetrahymena spp. and detergent-mediated lysis. SDS-PAGE analysis and shotgun proteomics revealed a number of VBNC cell specific proteins; one of these was 3-hydroxybutyrate dehydrogenase (BdhA), which is involved in the metabolism of poly 3-hydroxybutyrate inclusion bodies. A bdhA mutant showed an early loss of culturability and a dramatic decrease in viability as compared to the parent strain, and complementing the mutant with a functional bdhA gene restored the parent's strain phenotypes. In conclusion, VBNC L. pneumophila has a distinct morphology and physiology that varies according to the developmental stage and the environmental conditions used to produce such VBNC cells. VBNC cells have a different protein profile and morphology than the culturable cells, suggesting that this state constitutes a distinct differentiated form within the developmental cycle of L. pneumophila. BdhA seems to influence L. pneumophila survival and hence VBNC cell formation. Collectively, the results from this study provide a better understanding of L. pneumophila VBNC form and the factors influencing its formation.