Global cellular changes induced by Legionella pneumophila infection of bone marrow-derived macrophages

Abstract

The nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member Naip5 plays an essential role in restricting Legionella pneumophila growth inside primary macrophages. Upon interaction with bacterial flagellin, the intracellular receptor Naip5 forms a multi-protein complex, the inflammasome, which activation has a protective role against infection. The A/J mouse strain carries a Naip5 allele (Naip5(A/J)), which renders its macrophages susceptible to Legionella infection. However, Naip5(A/J) is still competent for inflammasome activation suggesting that an as yet unidentified signaling pathway located downstream of Naip5 and defective in Naip5(A/J) macrophages regulates macrophage defenses against Legionella. Therefore, transcriptional profiling experiments with macrophages from C57BL/6J mice (B6), and from congenic mice (BcA75) carrying the partial loss-of-function A/J-derived allele (Naip5(A/J)) on a B6 background, infected or not with wild-type L. pneumophila or flagellin-deficient mutant were carried out to identify genes regulated by flagellin and by Naip5. Both the Legionella infection per se and the presence of flagellin had very strong effects on transcriptional responses of macrophages, 4h following infection, including modulation of cellular pathways associated with inflammatory response and cell survival. On the other hand, the presence of wild type or partial loss of function allele (Naip5(A/J)) at Naip5 did not cause large effects on transcriptional responses of macrophages to infection. We also examined in L. pneumophila infected macrophages, the effect of Naip5 alleles on expression and phosphorylation of 524 phosphoproteins, kinases and phosphatases involved in cell proliferation, immune response, stress and apoptosis. Naip5 alleles had an effect on the TLR-Il1R signaling pathway, the cell cycle and the caveolin-mediated response to pathogen. The results of transcriptome and proteome analyses were organized into cellular pathways in macrophages that are modulated in response to Legionella infection.