Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts

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.

A Legionella pneumophila amylase is essential for intracellular replication in human macrophages and amoebae

“…pneumophila during post-exponential growth, most notably for the generation of the storage molecule, PHB 39 , 46 , 49 , and nutrient importers are important for intracellular growth of L . pneumophila 6 , 87 . Having large stores of PHB allows the organism to persist outside of the host for extended periods of time 51 .Transcriptomic studies in the human macrophage cell line THP-1 have demonstrated that expression of lamB is highest early in infection (8 hrs) rather than later (14 hrs), opposite of when the organism starts increasing consumption of glucose 46 , 88 .…”

Protozoan-Priming and Magnesium Conditioning Enhance Legionella pneumophila Dissemination and Monochloramine Resistance

“…To multiply within hosts, Lp requires a Dot/Icm type IVb secretion system to deliver more than 300 effector proteins (comprising nearly 10% of the Lp genome) directly into the host cells; this allows Lp to evade host defenses and establish replication vacuoles. , As repeated cycles of intracellular replication followed by dissemination are believed to be critical to Legionella persistence in the environment, this extensive catalogue of effector proteins is believed to reflect the diverse range of hosts that Legionella is capable of infecting. , It has been hypothesized that the interaction of Lp with environmental hosts is what led to the evolution of Lp ’s human virulence. − Despite the similar strategies used by Lp for infecting FLA and human macrophage, Lp ’s increased infectivity upon egress appears to be exclusive to passage through FLA . Key human virulence factors must therefore be produced by Lp in response to stimuli within FLA, a process we have previously coined protozoan-priming . However, the specific stimuli within FLA that contribute to the increased human virulence have yet to be elucidated.…”

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts byLegionella pneumophila

“…Within the LCV, bacterial metabolism and nutrition-based genetic regulation based in nutrient availability governs bacterial proliferation and differentiation (W. Eisenreich, Heesemann, Rudel, & Goebel, 2013; Wolfgang Eisenreich, Rudel, Heesemann, & Goebel, 2017; Fonseca & Swanson, 2014; Grubmuller, Schauer, Goebel, Fuchs, & Eisenreich, 2014; Häuslein et al, 2017; Lama, Drennan, Johnson, Rubenstein, & Cambronne, 2017; Manske & Hilbi, 2014; Oliva, Sahr, & Buchrieser, 2018; C. T. Price, Richards, & Abu Kwaik, 2014).…”