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Quantitative Proteomic Analysis of Extracellular Matrix Extracted from Mono- and Dual-Species Biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis

ABSTRACT: Background The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are associated with periodontitis and members of a complex microbial biofilm. The self-produced extracellular polymeric matrix (EPM) of biofilms generally consists of lipids, exopolysaccharides, nucleic acids and proteins. The proteinaceous components of the biofilm matrix play many roles in biofilm development and function. In this study the protein composition of the matrices in mono- and dual-species biofilms of F. nucleatum and P. gingivalis were analyzed. Methods The bacteria were grown in a static biofilm model. Extraction of EPM was performed by careful mechanical shearing and filtration. Initial tryptic digestion of EPMs was followed by liquid chromatography-tandem mass spectrometry to analyze the resulting peptide mixtures. Quantitative proteomic software MaxQuant was then employed for protein identifications and label-free quantitative (LFQ) analysis. Functional analyses of identified proteins were applied in classification and prediction of their possible roles in EPM. Results We identified 542 proteins in the matrix of F. nucleatum, 93 proteins in the matrix of P. gingivalis and 280 proteins in the dual-species biofilm matrix. Similarly, relative quantification based on LFQ intensity scores identified generally higher protein amounts in F. nucleatum and dual species EMPs, when compared to P. gingivalis EPM. Acetyl-CoA acetyltransferase, alkyl hydroperoxide reductase C22 protein, neutrophil-activating protein A, tryptophanase and glutamate dehydrogenase were the top five proteins in the matrix of F. nucleatum biofilm. The oxidoreductase NAD-specific glutamate dehydrogenase and the proteolytic and adhesive protein Lys-gingipain were most abundant in P. gingivalis EPM. In the dual species EPM, F. nucleatum contributed to four out of the five with glutamate dehydrogenase on top. NAD-specific dehydrogenase from P. gingivalis was among the top five. Functional analyses with respect to virulence for example, revealed the stress-induced protein yicC in F. nucleatum EPM, while hemagglutinin HagA and fimbriae minor component FimD were among the top 5 in P. gingivalis EPM. Conclusions We showed that the biofilm matrix of two important periodontal pathogens is abundant in proteins and nearly 70% of all EPM proteins originate from F. nucleatum when grown together with P. gingivalis. Outer membrane proteins, virulence related and oxidative stress proteins were among the most abundant proteins identified.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Fusobacterium Nucleatum Subsp. Nucleatum Atcc 25586 Porphyromonas Gingivalis Atcc 33277

SUBMITTER: Marwan Mansoor Ali Mohammed

LAB HEAD: Vidar Bakken

PROVIDER: PXD004888 | Pride | 2017-03-16

REPOSITORIES: Pride

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Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

Mohammed Marwan Mansoor Ali MMA Pettersen Veronika Kuchařová VK Nerland Audun H AH Wiker Harald G HG Bakken Vidar V

Anaerobe 20170309

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and seque ...[more]

PMID: 28285095

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Project description:Investigation of whole genome gene expression levels of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, S. mutans UA159 in an 24 h old culture. Additionally, whole genome gene expression level changes of S. mutans UA159 biofilm cells after co-cultivation with S. mitis ATCC 11843 were compared to its single species biofilm growth after 24 h. Aim: Demonstration of the usefulness of a five-species gene expression array. Multiple probes per gene enabled identification of single inter-species cross-hybridizing probes. The deletion of such probes lead almost not to the deletion of the whole gene. This was investigated and confirmed by a two-species biofilm expression analysis: The here described array was used for the identification of genes of S. mutans influenced by the presence of S. mitis. Materials and Methods: P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651,and S. mutans UA159 were grown in CDM/succrose or artificial saliva/galactose in a single-species culture for 24 h anaerobically resulting in biofilm structures or monolayers. Total RNA was isolated and used for microarray analysis. Probes were analysed for the presence of biological false positive signals caused by cross-hybridizing probes of one of the other species presented on the chip. Further, a simple procedure was developed for automatical identification and deletion of false positive signals caused by washing artefacts, resulting in a more reliable outcome. In the case of the S. mutans/S. mitis mixed-species biofilm, both species were cultured together for 24 h like previously described. The found gene regulations were verified by RT-PCR. Results: Experiments with cDNA from 24 h old single-species cultures allowed the identification of cross-species hybridizing probes on the array, which can be eliminated in mixed-species experimental settings without the need to exclude the whole genes from the analysis. Between 69 % and almost 100 % represented genomes on this array were found actively transcribed under the mono-species monolayer and biofilm conditions used here. S. mutans / S. mitis co-culture: Physiological investigations revealed an increase in S. mutans biofilm mass with a decrease in pH-value under the influence of S. mitis, thereby confirming previously published data. A stringent fold change cut-off of 2 (p<0.05) identified 19 S. mutans transcripts with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Many of the genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. Conclusions: Taken together, this new array allows transcriptome studies on multi-species oral biofilm interactions and could become an important asset in future oral biofilm and inhibitor/therapy studies. The chip study used pooled total RNA recovered from three biologically independent mono-species biofilms or adherent cells/monolayers of P. gingivalis W83, F. nucleatum DSMZ 25586, S. sanguinis SK36, A. actinomycetemcomitans HK1651, and S. mutans UA159. In the case of gene expression analysis of S. mutans/S.mitis biofilm structures compared to the single species biofilm of S. mutans three separate single and three separate two-species biofilm cultures were analysed. Each chip measured the expression level of all together 10186 genes (1883 genes of P. gingivalis W83, 1964 genes of F. nucleatum DSMZ 25586, 2244 genes of S. sanguinis SK36, 2168 genes of A. actinomycetemcomitans HK1651, 1927 genes of S. mutans UA159) with up to thirteen 60-mer probes per gene and with a three-fold technical redundancy.

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.

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