Abstract
Legionella pneumophila, the causative agent of Legionnaire’s disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of anti-apoptotic genes positively controlled by the transcriptional regulator NF-?B. Consistent with this finding, L. pneumophila triggered nuclear localization of NF-?B in human and mouse macrophage in a Dot/Icm-dependent manner. The mechanism of activation at low dose infections involved a signaling pathway that occurred independently of the TLR adaptor MyD88, and cytoplasmic sensor Nod1. In contrast, high MOI conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-?B. Inhibition of NF-?B translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one anti-apoptotic protein, PAI-2, host cell death increased in response to L. pneumophila infection, indicating that induction of anti-apoptotic genes is critical for host cell survival.
Keywords: time course, dose response
Overall design: To identify host cell genes that may modulate intracellular growth of L. pneumophila, we compared the transcriptional profile of U937 cells incubated with either WT L. pneumophila or a dotA- mutant lacking a functional Dot/Icm type IV translocator. The isolation of a homogenously infected cell population was a prerequisite for this study. Unlike other pathogens, L. pneumophila causes rapid Dot/Icm-dependent cytotoxicity at the high multiplicities of infection (MOI) that are required to infect the majority of cells in the monolayer. To bypass cytotoxicity, incubations unless indicated were performed at MOI=1 (1 bacterium per macrophage), which results in a approximately 30%the cultured cells having associated bacteria. To specifically analyze this population, U937 cells having associated bacteria were sorted away from the vast excess of uninfected cells using the GFP marker. Samples were collected 1 or 8 hours post infection, sorted for the cell population harboring GFP bacteria, and total RNA was isolated from these sorted populations. These two time points were chosen because the Dot+ strain initiates biogenesis of the replication vacuole at 1 hpi, with clear recruitment of ER-derived vesicles at this time point, and has completed one round of division by 8 hpi. In contrast, the type IV secretion-defective strain dotA-GFP strain fails to establish an ER-like vacuole and to replicate. To confirm that any changes in expression patterns were a result of a response to functions performed by the type IV translocation system, the mutant strain dimB- was included in this study. The dimB- mutant has an intact Dot/Icm translocator and shows proper formation of a replication vacuole, but is defective for intracellular growth and stalls after one intracellular doubling.
A total of 27 microarrays were used in this study. For each condition three independent microarrays were performed on cells isolated from three independent infections. The conditions were sorted U937 infected with WT, dimB, dotA at MOI=1 for 1 or 8 hours, as well as U937 cells infected with dotA at MOI=10. As control of gene expression changes due to sorting alone sorted uninfected U937 cells were included. No dye swaps were used. cDNA isolated from uninfected, unsorted U937 cells was always labeled with Cy3, and the sorted sample or high moi sample was always labeled with Cy5. The same reference total RNA preparation was used with each microarray experiment.
Less...