BioProject

Recently described microarray studies have defined the genes that are regulated in response to iron and heme levels in three isolates of Haemophilus influenzae. More...

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. Overall design: Three replicate cultures were grown in iron and heme depleted media for 90 minutes and samples taken for expression analyses. Fe and heme were supplemented to the remaining cultures and the cultures allowed to incubate an additional 20 minutes. Microarray analyses were used to determine changes in gene expression resulting from transfer from FeHm-deplete to FeHm-replete conditions. Less...