Dataset Information

Histone-like family of nucleoid structuring protein of A. actinomycetemcomitans promotes biofilm formation and alters protein dynamics of other species within multi-species biofilm

ABSTRACT: Aggregatibacter actinomycetemcomitans is a Gram-negative organism, strongly associated with aggressive forms of periodontitis. An important virulence property of A. actinomycetemcomitans is its ability to form tenacious biofilms that can attach to abiotic as well as biotic surfaces. The histone-like (H-NS) family of nucleoid structuring proteins act as transcriptional silencers in many Gram-negative bacteria. To evaluate the role of H-NS in A. actinomycetemcomitans, hns mutant derivatives of the serotype a strain D7S were generated. Characteristics of the hns mutant phenotype included shorter and fewer pili, which were accompanied by substantially lower biofilm formation relative to the wild type. In accordance with this observation, the D7S hns mutant exhibited significantly reduced growth within a seven-species biofilm model. However, no apparent difference was observed regarding the numbers and proportions of the remaining six species in the biofilm regardless co-cultivated with D7S hns or its parental strain. Proteomics analysis of the strains grown in monocultures confirmed the role of H-NS as a repressor of gene expression in A. actinomycetemcomitans. Interestingly, proteomics analysis of the multispecies biofilms indicated that the A. actinomycetemcomitans wildtype and hns mutant imposed different regulatory effects on the pattern of protein expression in the other species, i.e. mainly Streptococcus spp., Fusobacterium nucleatum, and Veillonella dispar. Gene ontology analysis revealed that a large portion of the differentially regulated proteins was related to translational activity. Taken together, our data suggest that, apart from being a negative regulator of gene expression in A. actinomycetemcomitans, H-NS promotes biofilm formation and may be an important factor for survival of this species within a multispecies biofilm.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Aggregatibacter Aphrophilus Streptococcus Mutans Streptococcus Oralis Fusobacterium Nucleatum Veillonella Dispar Candida Albicans (yeast) Actinomyces Oris Aggregatibacter Actinomycetemcomitans (actinobacillus Actinomycetemcomitans) (haemophilus Actinomycetemcomitans)

TISSUE(S): Biofilm

SUBMITTER: Kai Bao

LAB HEAD: Nagihan Bostanci

PROVIDER: PXD008444 | Pride | 2018-06-05

REPOSITORIES: Pride

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Aggregatibacter actinomycetemcomitans H-NS promotes biofilm formation and alters protein dynamics of other species within a polymicrobial oral biofilm.

Bao Kai K Bostanci Nagihan N Thurnheer Thomas T Grossmann Jonas J Wolski Witold E WE Thay Bernard B Belibasakis Georgios N GN Oscarsson Jan J

NPJ biofilms and microbiomes 20180522

Aggregatibacter actinomycetemcomitans is a Gram-negative organism, strongly associated with aggressive forms of periodontitis. An important virulence property of A. actinomycetemcomitans is its ability to form tenacious biofilms that can attach to abiotic as well as biotic surfaces. The histone-like (H-NS) family of nucleoid-structuring proteins act as transcriptional silencers in many Gram-negative bacteria. To evaluate the role of H-NS in A. actinomycetemcomitans, hns</ ...[more]

PMID: 29844920

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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: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.

Project description:Multispecies biofilms are the predominant form of bacterial growth in natural and human-associated environments. Although the pathways involved in monospecies biofilm have been well characterized, less is known about the metabolic pathways and emergent traits of a multispecies biofilm community. Here, we performed a transcriptome survey of the developmental stages of a 3-species biofilm community and combined it with quantitative imaging and growth experiments. We report the remodelling of central metabolism of two of the three species in this community. Specifically, we observed an increase in the expression of genes associated with glycolysis and pentose phosphate pathways in K. pneumoniae. Similarly, a decrease in the expression of the same pathways in P. protegens was observed along with an increase in expression of glyoxalate cycle genes when grown as a mixed species biofilm, suggesting reorganisation of metabolic pathways and metabolite sharing for the community biofilms. To test the possibility of cross-feeding for the community, planktonic growth experiments revealed that both the Pseudomonads grew well in TCA cycle intermediates, while K. pneumoniae grew poorly when given those carbon sources. Despite this poor growth in mono-culture, K. pneumoniae was still the dominant species in mixed species biofilms cultivated in TCA intermediates as the sole source of carbon. The biofilm growth data, combined with the transcriptomics data, suggests there is reorganisation of metabolism for the community members and may allow for cross-feeding that allows K. pneumoniae to dominate the community. We also demonstrated that sdsA1 of P. aeruginosa was induced upon exposure to the surfactant SDS and that this gene was essential in protecting mono and mixed species biofilms from surfactant stress. This also suggests that the community members can share defence mechanisms. Overall, this study describes a comprehensive transcriptomics level investigation of shared resources, metabolites and stress defence that may underpin the emergent properties of mixed species biofilm communities.

Dataset Information

Histone-like family of nucleoid structuring protein of A. actinomycetemcomitans promotes biofilm formation and alters protein dynamics of other species within multi-species biofilm

Publications

<i>Aggregatibacter actinomycetemcomitans</i> H-NS promotes biofilm formation and alters protein dynamics of other species within a polymicrobial oral biofilm.

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