The influence of phenotype on the stress resistance of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an aquatic intracellular organism capable of replicating within both amoebae and human phagocytic cells. A comprehensive study has been undertaken to examine the hypothesis that intracellular replication of L. pneumophila induces enhanced resistance to external stress stimuli relative to that of in vitro grown legionellae.Microscopical studies have shown that L. pneumophila grown in YE broth consistently develop a rod-shaped morphology and are non-motile. In contrast, L. pneumophila grown within Acanthamoeba polyphaga or U937 monocytes exhibit a smaller, rounded morphology and are highly motile. After ca. 72 h post lysis, the intracellular bacteria adopted a morphology similar to that of broth grown legionellae in stationary phase. These bacteria were termed 'aged'.Time-kill assays have shown that L. pneumophila grown within A. polyphaga or U937 monocytes are more resistant than broth grown legionellae to a number of different stress conditions including exposure to the antibiotics traditionally used in the treatment of Legionnaires' disease, elevated temperatures associated with water treatment processes, mechanical stress and starvation. 'Ageing' of intracellular L. pneumophila before exposure to the stress stimuli, resulted in a marked loss of stress resistance.To study the physiological basis of the increased stress resistance of intracellular grown L. pneumophila, a preliminary study of the surface properties of the variously grown legionellae was undertaken. The outer membrane protein (OMP) profiles of the L. pneumophila were prepared using sarkosyl and analysed by SDS-PAGE. Proteinase K digestion of the outer membrane (OM) was employed to prepare the LPS layer. The preliminary comparative study of the L. pneumophila OM has established that the mode of growth influences both the OMP profile and the LPS layer. In particular, intra-amoebic grown L. pneumophila possesses a novel protein of 15 kDa and intra-monocytic grown legionellae one of 24 kDa, both of which are lost upon 'ageing'.The results of the project have shown a link between the presence of novel sarkosyl insoluble OMPs, bacterial morphology and enhanced resistance of the intracellular grown L. pneumophila to external stress stimuli. It could, therefore, be suggested that L. pneumophila undergo a prior adaptation to stress conditions during intracellular replication. Upon 'ageing' of intracellular grown legionellae the novel insoluble OMPs were lost with a concomitant change in morphology and loss in resistance to external stress stimuli.The findings of this work have practical implications with respect to the clinical treatment of Legionnaires' disease and the eradication of the causative agent from water systems.