Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of gene expression in Porphyromonas gingivalis hemH and ihtB deletion mutant strains grown under heme- and iron-replete conditions (RNA-seq analysis)

ABSTRACT: Porphyromonas gingivalis is a keystone pathogen responsible for oral microbiome dysbiosis, the development of periodontal disease, and may be involved in the development of systemic comorbidities. As a heme auxotroph, P. gingivalis relies on host hemoproteins as its primary sources of heme and iron. Although heme acquisition in P. gingivalis is relatively well characterized, considerably less is known about its pathways for iron uptake and the regulation of iron and heme homeostasis. P. gingivalis encodes four enzymes associated with the heme biosynthesis pathway, namely HemD, HemN, HemG, and HemH. HemH is a ferrochelatase responsible for the final step of heme biosynthesis, catalyzing the insertion of ferrous iron (Fe²⁺) into protoporphyrin IX. In addition, P. gingivalis expresses the Iht system, which plays a role in iron/heme utilization. One of its components, IhtB, is a heme-binding protein homologous to CbiK cobaltochelatases. Based on this homology, IhtB is hypothesized to function as a reverse ferrochelatase, facilitating the removal of iron from heme and thereby contributing to iron acquisition. This study aimed to investigate the effect of ihtB and hemH gene deletions on P. gingivalis gene expression under heme- and iron-replete conditions, to elucidate the roles of IhtB and HemH chelatases in heme and iron homeostasis.

INSTRUMENT(S): Illumina NovaSeq X

ORGANISM(S): Porphyromonas gingivalis

SUBMITTER: Michał Śmiga

PROVIDER: E-MTAB-16135 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Porphyromonas gingivalis is a major pathogen associated with the microbial biofilm-mediated disease chronic periodontitis. P. gingivalis has an obligate requirement for iron and protoporphyrin IX which it satisfies by transporting heme and iron liberated from the human host. The level of cellular iron in P. gingivalis affects the expression of a distinct iron-associated regulon of 64 genes and low iron invokes an iron sparing response. Iron homeostasis is usually mediated in Gram-negative bacteria at the transcriptional level by the Ferric Uptake Regulator (Fur). There is a single predicted P. gingivalis Fur superfamily orthologue named Har (heme associated regulator) that lacks the conserved metal binding residues found in other Fur orthologues. We show that Har binds both heme and ferrous iron resulting in a conformational change in the protein. Har was unable to complement the Escherichia coli H1780 fur mutant and there was no change in cellular metal content in a P. gingivalis Har mutant compared with the wild-type. The Har regulon of 44 genes is not predicted to play a role in iron homeostasis. Together these data indicated that Har does not regulate iron homeostasis in P. gingivalis. However, Har was required for heme-responsive biofilm development and its regulon overlapped P. gingivalis regulons previously identified after growth in heme limitation or as a homotypic biofilm. P. gingivalis is unique as an iron-dependent Gram-negative bacterium with a single heme-binding Fur superfamily orthologue, Har, that does not regulate iron homeostasis. Paired samples were compared on the same microarray using a two-colour system. A total of 6 paired microarray hybridizations were performed representing 6 biological replicates, where a balanced dye design was used, with the overall analysis including three microarrays where P. gingivalis 33277 samples were labeled with Cy3 and the paired ECR455 samples were labeled with Cy5 and three other microarrays where samples were labeled with the opposite combination of fluorophores.

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 M-bM-^@M-^\iron and heme regulated genes are upregulated, and important, during clinical infectionM-bM-^@M-^]. 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. 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.

Dataset Information

Analysis of gene expression in Porphyromonas gingivalis hemH and ihtB deletion mutant strains grown under heme- and iron-replete conditions (RNA-seq analysis)

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