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Legionella pneumophila 6S RNA optimizes intracellular multiplication.

ABSTRACT: Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which 6 were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed in postexponential phase, and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of sigma(70)-containing RNA polymerase. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA, as well as many genes involved in acquisition of nutrients and genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.

SUBMITTER: Faucher SP

PROVIDER: S-EPMC2867745 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Legionella pneumophila 6S RNA optimizes intracellular multiplication.

Faucher Sébastien P SP Friedlander Gilgi G Livny Jonathan J Margalit Hanah H Shuman Howard A HA

Proceedings of the National Academy of Sciences of the United States of America 20100405 16

Legionella pneumophila is a Gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to identify sRNAs expressed by L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the ...[more]

PMID: 20368425

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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. The aim of this work was to study the gene expression profile of Legionella during infection of macrophages infected at a low multiplicity of infection (MOI). SCOTS is a method that allows amplification of small amounts of bacterial RNA from infected host cells, while discarding host cell transcripts and ribosomal RNA. Before infection, macrophages derived from the THP-1 monocyte cell line were pre-treated with antibodies against the L. pneumophila major outer membrane protein, which increases the efficiency of bacterial entry into host cells . After 2 hours of infection, the macrophages were washed and treated with gentamicin for 1 hour, to synchronize the infection and kill extracellular bacteria, and cells were washed 3 times and fresh medium was added. Samples for the first time point (T0) were collected after the gentamicin treatment. Samples were also collected after 6h (T6) and 18h (T18). Samples from all conditions, including growth in AYE broth to exponential (E) or post exponential (PE) phase, were treated with three consecutive rounds of SCOTS and the resulting cDNA was labeled and hybridized to the microarray slides. As a reference channel, labeled gDNA was also hybridized. For each condition studied, three independent biological replicates and two technical replicates were analyzed, resulting in six replicates for each condition. The data was background subtracted and normalized by calculating the contribution of each spot to the total intensity and the ratio to gDNA was recorded. A one tailed student's T test was used for statistical analysis and the ratio between test conditions (PE, T0, T6 and T18) and the control conditions (E phase or T0) was calculated.

Project description:Vacuolar bacterial pathogens are sheltered within unique membrane-bound organelles that expand over time to support bacterial replication. These compartments sequester bacterial molecules away from host cytosolic immunosurveillance pathways that induce antimicrobial responses. The mechanisms by which the human pulmonary pathogen Legionella pneumophila maintains niche homeostasis are poorly understood. We uncovered that the Legionella-containing vacuole (LCV) required a sustained supply of host lipids during expansion. Lipids shortage resulted in LCV rupture and initiation of a host cell death response, whereas excess of host lipids increased LCVs size and housing capacity. We found that lipids uptake from serum and de novo lipogenesis are distinct redundant supply mechanisms for membrane biogenesis in Legionella-infected macrophages. During infection, the metabolic checkpoint kinase Mechanistic Target of Rapamycin (MTOR) controlled lipogenesis through the Serum Response Element Binding Protein 1 and 2 (SREBP1/2) transcription factors. In Legionella-infected macrophages a host-driven response that required the Toll-like receptors (TLRs) adaptor protein Myeloid differentiation primary response gene 88 (Myd88) dampened MTOR signaling which in turn destabilized LCVs under serum starvation. Inactivation of the host MTOR-suppression pathway revealed that L. pneumophila sustained MTOR signaling throughout its intracellular infection cycle by a process that required the upstream regulator Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and one or more Dot/Icm effector proteins. Legionella-sustained MTOR signaling facilitated LCV expansion and inhibition of the PI3K-MTOR-SREPB1/2 axis through pharmacological or genetic interference or by activation of the host MTOR-suppression response destabilized expanding LCVs, which in turn triggered cell death of infected macrophages. Our work identified a host metabolic requirement for LCV homeostasis and demonstrated that L. pneumophila has evolved to manipulate MTOR-dependent lipogenesis for optimal intracellular replication.

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Legionella pneumophila 6S RNA optimizes intracellular multiplication.

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Legionella pneumophila 6S RNA optimizes intracellular multiplication.

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