Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila

Abstract

SummaryAdaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii . The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro , may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA ( s 28 ) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.

FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

“…The rpoN gene of L. pneumophila Corby was nearly constitutively expressed and thus in good agreement with the results published for strain Paris (Brüggemann et al 2006;AlbertWeissenberger et al 2010). Therefore, rpoN served as loading control for the RT-PCR analysis.…”

“…Experiments were done with L. pneumophila Sg1 strain Corby (Jepras et al 1985) and L. pneumophila Sg1 strain Paris (Cazalet et al 2004) and their isogenic ΔrpoN, ΔfleQ and ΔfliA mutant strains (Heuner et al 2002;Jacobi et al 2004;Albert-Weissenberger et al 2010;Brüggemann et al 2006). Further Corby strains used in this study were ΔflaA, ΔmotA and ΔfliD (Dietrich 2000;Heuner and Albert-Weissenberger 2008).…”

“…pKH3 contained the flaA gene which was disrupted by a Km r cassette. The insert was amplified by using the primers M13U and The A. castellanii infection assay for in vivo growth of Lp strains Corby and Paris and their derivatives was done as described before (Brüggemann et al 2006). In brief, 3-day-old cultures of A. castellanii were washed in AC buffer [PYG 712 medium without proteosepeptone, glucose and yeast extract], adjusted to 1 x 10 5 cells per ml and incubated in 24-well plates for 2 hours at 37°C and 5% CO 2 .…”

“…In this phase L. pneumophila expresses many virulence-associated traits, including the flagellum (Brüggemann et al 2006;Byrne and Swanson 1998;Heuner and AlbertWeissenberger 2008;Molofsky et al 2005;Sauer et al 2005a;Sauer et al 2005b) Hammer et al 2002;Molofsky and Swanson 2004). It is believed that LetA/LetS responds to the alarmone (p)ppGpp and the intracellular concentration of (p)ppGpp is regulated by RelA and SpoT (Dalebroux et al 2010a;Edwards et al 2009;Dalebroux et al 2009;Hammer and Swanson 1999;Hammer et al 2002;Zusman et al 2002).…”

“…The alternative 28 factor FliA (class III gene) directly controls the expression of the flagellar class IV genes (e.g. flaA) (Albert-Weissenberger et al 2010;Brüggemann et al 2006;Jacobi et al 2004;Heuner and Albert-Weissenberger 2008). It is known that L. pneumophila rpoN, fleQ, fliA and flaA mutant strains are non-flagellated and non-motile (Heuner et al 1995;Heuner et al 2002;Heuner et al 1997;Jacobi et al 2004;Molofsky et al 2005).…”