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Identification of a FimA protein in a crude lysate SDS-PAGE gel of a P. gingivalis heme-uptake mutant

ABSTRACT: Porphyromonas gingivalis is one of the main etiological agents responsible for initiating and progressing periodontal diseases in humans. P. gingivalis does not synthesize heme, and it uses host hemoproteins as a source of this molecule. The main heme uptake mechanism utilized by P. gingivalis is the Hmu system composed of six proteins where HmuY (hemophore-like protein) and HmuR (TonB-dependant outer membrane heme receptor) play a crucial role in heme supply. The second heme uptake mechanism is the Hus system composed of four proteins, with HusA (hemophore-like protein) and HusB (TonB-dependant outer membrane receptor), having a supporting role in heme uptake. Heme and iron starvation influence the P. gingivalis phenotype, therefore the aim was to determine the influence of deletion of HmuY and HmuR (ΔhmuYR strain) or HusA and HusB (ΔhusAB strain) encoding genes on P. gingivalis phenotype including the impact on protein production in response to the analyzed deletion. The mutant strains were constructed in wild-type A7436 P. gingivalis strain. Bacteria were grown in basal medium (BM) supplemented with 7.7 µM heme. After analysis of bacteria lysates with SDS-PAGE and CBB G-250 staining, in ΔhmuYR a protein (~45 kDa) was detected produced in higher amounts, than in the wild-type and the ΔhusAB strains. The gel band of interest, as well as its corresponding sections in the WT lanes were excised for bottom-up LC-MS analysis, and the protein was identified as FimA.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Porphyromonas Gingivalis A7436

TISSUE(S): Cell Lysate

SUBMITTER: Michał Tracz

LAB HEAD: Michal Tracz

PROVIDER: PXD054703 | Pride | 2024-12-28

REPOSITORIES: pride

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			Action	DRS
	240510_ZBCH_MSM_TO2_1.mgf	Mgf
	240510_ZBCH_MSM_TO2_1.mzid.gz	Mzid
	240510_ZBCH_MSM_TO2_1.raw.zip	Raw
	240510_ZBCH_MSM_TO2_1_IA_final_protein.csv	Csv
	240510_ZBCH_MSM_TO2_2.mgf	Mgf

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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.

Dataset Information

Identification of a FimA protein in a crude lysate SDS-PAGE gel of a P. gingivalis heme-uptake mutant

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