Project description:The RNA binding protein CsrA is the master regulator of the bi-phasic life cycle of Legionella pneumophila governing virulence expression in this intracellular pathogen. The goal of the study was to use deep sequencing of RNA enriched by co-immunoprecipitation with epitope tagged CsrA to identify CsrA-associated transcripts at the genome level. We found 478 mRNAs or non-coding RNAs to be targets of CsrA. Among those major regulators including FleQ, the regulator of flagella expression, LqsR, the regulator of quorum sensing and RpoS implicated in stress response were identified. The expression of over 40 type IV secreted effector proteins important for intracellular survival and virulence are under the control of CsrA. Combined with transcriptomics, whole shotgun proteomics of a wild type and a CsrA mutant strain and functional analyses of several CsrA-targeted RNAs we identified the first riboswitch in L. pneumophila, a thiamine pyrophosphate riboswitch, and discovered a new mode of regulation by CsrA that allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Our results further underline the indispensable role of CsrA in the life cycle of L. pneumophila and provide new insights into its regulatory roles and mechanisms.
Project description:The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. In this dataset we identified proteins regulated by CsrA in the human bacterial pathogen Legionella pneumophila by labelfree shotgun proteomics. WT and ∆csrA bacteria were grown to exponential phase in three biological replicates. Bacteria were lysed by sonication and extracted proteins were reducted, alkylated and digested with trypsin. The resulting peptide mixtures were analyzed by LC-MS/MS and proteins were identified by the MaxQuant software. In total of 1,448 proteins were quantified by the MaxLFQ algorithm in a labelfree approach. Cluster analysis showed that expression of almost all proteins was affected by the mutation of CsrA as about half of the identified proteins were up and half were down regulated. About 15% of the proteome (238 proteins) was strongly affected with 132 proteins significantly up and 109 significantly down regulated dependent on CsrA.
Project description: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
Project description:Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoan, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known virulence determinant used by L. pneumophila to infect host cells is a Type IVb translocation system named Icm/Dot, which is used to modify the host cell functions to the benefit of the bacteria. To date the Icm/Dot systeme is known to translocate more than 100 effectors. While the transcriptional response of Legionella to the intracellular environement of A. castelannii as already been investigated, much less is known of how Legionella reacts transcriptionnally inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth and during infection of human cultured macrophages by using microarray and a RNA amplification procedure called SCOTS to allow for the study of conditions of low bacterial loads. Among the genes induced intracellularly are those involved in amino acid synthesis pathway leading to L-arginine, L-histidne and L-proline as well as many transport system involved in amino acid and iron uptake. The Icm/Dot systems is not differentially expressed inside cells compare to the E phase control but the effectors are strongly induced. The intracellular transcriptome was further used to identify putative new Icm/Dot effectors and translocation was show to occur for 3 of them. This study provides a comprehensive view of how L. pneumophila react to the human macrophages intracellular environment.
Project description:Legionella pneumophila cells were harvested during exponential growth (RP) and stationary growth (TP). VBNC cells were also anylzed. Protein subfractions were studied.
Project description:Comparaison of the transcriptional profile of mutant of the lpp1663 gene and the parent strain Legionella pneumophila strain Paris. lpp1663 codes for a protein with a ProQ domain (PFAM PF04352). A mutant of this gene was obtained by deleting the lpp1663 ORF and replacing it with a gene confering resistance to kanamycin. Transcriptional profiling was done on cultures grown to exponential phase (OD=0.8). Three independent biological replicates were analysed.