Transcription profiling of Listeria monocytogenes intracellularly replicating in mouse macrophages activated by IFN-gamma
ABSTRACT: The present study show the impact of macrophages activation by IFN-Gamma on the gene regulation in intracellularly replicating L. monocytogenes and provide an overview about additionally adaption mechanisms of L. monocytogenes to immune response.
INSTRUMENT(S): Generation III Array Scanner [Amersham]
Project description:We infected wild type L. monocytogenes EGD-e (1) and its isogenic deltahlydeltaplcA (2) (lacking the ability to breach the vacuolar compartment of host cells following uptake) mutant strain to human intestinal epithelial cell line (Caco-2) with an MOI of 100 and 500 respectively. Bacterial total RNA was isolated at 1 h (deltahlydeltaplcA) and 4 h (EGD-e) post infection, reverse transcribed, hybridised to whole genome microarray and microarray data was analysed as described previously (3) 1. Glaser et al. 2001. Comparative genomics of Listeria species. Science 294:849-852. 2. Paschen et al. 2000. Human dendritic cells infected by Listeria monocytogenes: induction of maturation, requirements for phagolysosomal escape and antigen presentation capacity. Eur.J.Immunol. 30:3447-3456. 3. Chatterjee et al. 2006. Intracellular gene expression profile of Listeria monocytogenes. Infect.Immun. 74:1323-1338.
Project description:Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes (Lm) expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500nt) isolated from extracellularly growing bacteria and from Lm-infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here we report on the discovery of 150 regulatory RNAs of which 71 have never been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by Northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50 for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNAs and the absence of sRNA loci in genomes of naturally-occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.
Project description:L. monocytogenes EGD-e (Glaser et al., 2001) was grown in Brain Hearth Infusion Broth (Difco) with shaking (200 rpm, New Brunswick C24KC) using 10 ml culture medium in 100 ml conical flasks. An exponentially growing culture was used to inoculate 100 ml of fresh pre-warmed BHI broth in a 500 ml flask. This culture was incubated at 48°C (GFL Type 1083, 60 % shaking speed) and samples for RNA extraction were taken after 3, 10, 20, and 40 min.
Project description:P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes serotypes (1/2a EGD-e, 4a L99, 4b CLIP80459 and 4b F2365) were infected to the P388D1 cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 µg/ml gentamicin. The medium of the plates (containing 20 µg/ml gentamicin) infected with L. monocytogenes serotypes were replaced after 2 h post infection with fresh medium containing 50 µg/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection.
Project description:We analyzed the transcriptional signatures of mouse bone marrow-derived macrophages (BMDM) at different times after infection with promastigotes of the protozoan parasite Leishmania major. GeneChip Mouse Gene 1.0 ST arrays were used to analyse global changes in gene transcripts to generate a pool of genes that was statistically significant (p-value < 0.05) and a fold change cut-off of 1.5 in at least one of the five infected samples versus non-infected samples. Three independent biological replicates for each biological condition (Non infected "NI", infected with lived parasite "P" or "heat inactivated parasite "Kp") at 1h (T1), 3(T3), 6(T6), 12 (T12) and 24hours (T24) were performed. The Non infected (NI) at 0 hour (T0) biological condition were used as an additional control.
Project description:The transcription factor STAT1 is essential for interferon- (IFN) mediated protective immunity in humans and mice. Two splice isoforms of STAT1, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. Dimers of STAT1β are therefore considered transcriptionally inactive and potential competitive inhibitors of STAT1α. Contrasting this view, generation and analysis of mice deficient for either STAT1α or STAT1β demonstrated transcriptional activity of the STAT1β isoform and its enhancement of innate immunity. Gene expression profiling in primary cells revealed overlapping, but also non-redundant and gene-specific activities of STAT1α and STAT1β in response to IFNγ. Consistently, both isoforms mediated protective, IFNγ-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiency. In contrast, STAT1α and STAT1β were largely redundant for transcriptional responses to IFNα/β and for IFNα/β-dependent antiviral activity. Collectively, our data shed new light on how STAT1 isoforms contribute to antimicrobial immunity. We treated macrophages of Stat1 delta alpha and Stat1 delta beta isoforms as well as Stat1 KO and Stat1 Wt mice with IFN gamma and Listeria to reveal differences in gene expression between isoforms and the two control genotypes.
Project description:Briefly, over night cultures of L. monocytogenes EGD-e cells (wild-type and sigma B mutant) were grown in brain heart infusion (BHI) broth (Difco). Bacteria were diluted 1:50 in 20 ml fresh BHI and incubated using a 100 ml Erlenmeyer flask at 37 °C with shaking (180 rpm, Unitron [Infors]). Bacterial cells were harvested at OD600 1.0 (3 h), OD600 2.0 (4 h), OD600 2.5 (8 h) OD600 2.5 (16 h) using RNAprotect (Qiagen) according to manufacturer.
Project description:Bacteria were grown in BHI until OD600 1.0 (late exponential phase) and RNA stabilized using the RNAprotectTM bacteria reagent (QIAGEN) according to the manufacturer recommendations. For each strain, RNA extraction has been carried out on three independent biological samples. Briefly, bacterial total RNA was isolated, reverse transcribed, hybridised to whole genome microarray and microarray data was analysed as described previously (Chatterjee, 2006). Hybridizations were performed with cDNAs from EGD-e versus the over-expressing strain (EGD-e+pPXrnhB).