Project description:Transcriptome analysis of NTHi 86-028NPrpsL, NTHi 86-028NPrpsL∆fur, and NTHi 86-028NPrpsL∆fur(pT-fur) strains Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the normal human nasopharyngeal flora, yet also an opportunistic pathogen of the upper and lower respiratory tracts. Changes in gene expression patterns in response to host microenvironments are likely critical for survival. One such system of gene regulation is the ability to carefully regulate iron uptake. A central regulatory system that controls iron uptake, mediated by the ferric uptake regulator Fur, is present in multiple bacteria, including NTHi. To understand the regulation of iron homeostasis in NTHi, fur was deleted in the NTHi strain 86-028NPrpsL. Using RNA-Seq, we identified both protein-encoding and small RNA genes whose expression was repressed or activated by Fur. Overall design: These data comprise transcriptional anaylses of an rpsL mutant of 86-028NP, an isogenic fur mutant of 86-028NPrpsL and a complemented fur mutant strain. All strains were grown in defined medium containing 10 µg/ml human hemoglobin to mid-log phase. Cells were then harvested and RNA extracted. A total of three biological replicates were generated for these analyses.
Project description:Transcriptome analysis of NTHi 86-028NPrpsL, NTHi 86-028NPrpsL∆fur, and NTHi 86-028NPrpsL∆fur(pT-fur) strains Nontypeable Haemophilus influenzae (NTHi) is a commensal microorganism of the normal human nasopharyngeal flora, yet also an opportunistic pathogen of the upper and lower respiratory tracts. Changes in gene expression patterns in response to host microenvironments are likely critical for survival. One such system of gene regulation is the ability to carefully regulate iron uptake. A central regulatory system that controls iron uptake, mediated by the ferric uptake regulator Fur, is present in multiple bacteria, including NTHi. To understand the regulation of iron homeostasis in NTHi, fur was deleted in the NTHi strain 86-028NPrpsL. Using RNA-Seq, we identified both protein-encoding and small RNA genes whose expression was repressed or activated by Fur. Overall design: These data comprise transcriptional anaylses of an rpsL mutant of 86-028NP, an isogenic fur mutant of 86-028NPrpsL and a complemented fur mutant strain. All strains were grown in defined medium containing 10 µg/ml human hemoglobin to mid-log phase. Cells were then harvested and RNA extracted. A total of three biological replicates were generated for these analyses. Analysis of transcriptomes using the Illumina HiSeq 2000 of three strains of nontypeable Haemophilus influenzae which include NTHi 86-028NPrpsL, NTHi 86-028NPrpsL∆fur, and NTHi 86-028NPrpsL∆fur(pT-fur) strains. For each strain three biological replicates were analyzed
Project description:Recently described microarray studies have defined the genes that are regulated in response to iron and heme levels in three isolates of Haemophilus influenzae. Comparison of the data sets have allowed us to develop a putative core iron/heme modulon. Included in the core modulon are thirty seven genes that are preferentially expressed under iron/heme limitation. The majority of these genes are involved with iron and or heme acquisition such as hitA, tonB, exbD, exbB, hgpB, hgpC and the hxu and tbp operons. In addition, we identified several other loci with roles potentially related to iron and heme metabolism such as protection against oxidative stress, iron and heme storage and detoxification and biofilm formation. In this report, we describe the further definition of the core iron/heme modulon following transcriptomic analysis of H. influenzae strains 86028NP and R2846. With the defined core, we then test our original hypothesis that “iron and heme regulated genes are upregulated, and important, during clinical infection”. The in vivo expression profiles of the core genes were determined following inoculation of select isolates into the bullae of chinchillas. Isolates selected included 86028NP and a previously un-characterized isolate HI1722. To facilitate studies with the latter isolate, we sequenced the genome and annotated the identified CDS to ensure that oligonucleotides selected for Q-PCR would correctly target each gene of interest. The results from the in vitro studies show that the operons identified as core are actively upregulated in the chinchilla ear during otitis media. For isolate 86-028NP, 70% of the operons were significantly upregulated while isolate NTHI1722 had 100% of the operons upregulated.