Abstract
It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term “protozoan-priming.” We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.
Highlights
Legionella pneumophila (L.p.) is a Gram-negative bacterium predominantly associated with freshwater environments
WipA, which was recently reported to harbor tyrosine phosphatase activity had been previously determined dispensable for intracellular survival of L.p. in both macrophage and amoebae (Ninio et al, 2005; Pinotsis and Waksman, 2017)
These results suggested that each of these loci were critical for survival in particular host cell types (A.c., Chinese Hamster ovary (CHO) FcγRII) while remaining completely dispensable in another (J774A.1)
Summary
Legionella pneumophila (L.p.) is a Gram-negative bacterium predominantly associated with freshwater environments. Free-living bacteria persist in water, yet replication is restricted to the confines of host protozoan cells (amoebae), where the bacterium is a facultative intracellular parasite (Fields, 1996; Abu Kwaik et al, 1998). In order to survive and replicate in eukaryotic cells, L.p. requires a specialized type IVb secretion system termed Dot/Icm (Marra et al, 1992; Berger and Isberg, 1993; Segal and Shuman, 1997). This multi-protein secretion apparatus functions to deliver up to 300 effector proteins into the host-cell (Zhu et al, 2011). Strategies adopted for survival in protozoa directly translated to efficient replication in the hostile environment encountered within the macrophage of the lung
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