Unknown,Transcriptomics,Genomics,Proteomics

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Stage-Specific Alterations in the Transcriptome of Lyme Disease Spirochetes During Tick Feeding and Following Mammalian Host-Adaptation

ABSTRACT: To compare the transcriptional profiles of Borrelia burgdorferi (Bb) during acquisition (fed larvae), transmission (fed nymphs) and in a mammalian host-like environment (dialysis membrane chambers [DMC]).RNA was isolated from an infected tick (Ixodus scapularis) samples, DMC and in vitro cultures. Array hybridization was performed with 7 RNA samples (from 5 biological replicates) obtained from fed nymphs and 10 RNA samples (from 6 biological replicates) obtained from fed larvae. Mammalian host-adapted spirochetes were generated by growth in DMCs. 4 arrays (2 biological replicates) were performed with DMC samples and in vitro-cultivated (temperature-shifted) samples.RNA was labeled with Cy3and as an internal standard for array hybridization, B. burgdorferi genomic DNA was labeled with Cy5 hybridized to Bb 70-mer slide arrays.Signal was acquired on a GenePix scanner. Background subtracted intensities obtained for each gene were normalized to one reference array using Microsoft Excel. Normalized data sets were further refined using GeneSpring software. A gene was scored as expressed if pixel intensity was >75 and was detected in a majority of arrays for given condition. Differentially expressed genes were identified as those with a fold change >2 at P<0.02 through gene-by-gene comparison of normalized expression data sets using an unpaired t-test within GeneSpring GX12.5.

INSTRUMENT(S): GenePix 4000B

ORGANISM(S): Borrelia burgdorferi

SUBMITTER: Radha Iyer

PROVIDER: E-MTAB-2957 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, ostensibly is required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN, and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN, and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN, or rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS, therefore providing additional avenues for future research into the genetic determinants contributing to virulence expression by B. burgdorferi. Keywords: gene profiling 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoS mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rpoN mutant: 5 slides, 10 hybridizations (including dyeswaps), three biological replicates 297 (wt) vs rrp2 mutant: 6 slides, 12 hybridizations (including dyeswaps), three biological replicates 5A18 (wt) vs XY424.3 (rrp2 mutant): 5 slides, 10 hybridizations (including dyeswaps), three biological replicates

Dataset Information

Stage-Specific Alterations in the Transcriptome of Lyme Disease Spirochetes During Tick Feeding and Following Mammalian Host-Adaptation

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