Dataset Information

Bacterial symbionts use a type VI secretion system for fitness increase against pathobionts

ABSTRACT: The Type VI Secretion System (T6SS) in bacteria is a versatile mechanism that facilitates protein transport into neighboring cells and can act as an antibacterial weapon by eliminating competing organisms in the vicinity. The objective of this study was to characterize the T6SS in Aggregatibacter aphrophilus and assess its antimicrobial capabilities through competition with Aggregatibacter actinomycetemcomitans in a multispecies biofilm. The proteomic analysis consisted of two parts, referred to as monospecies biofilm and multispecies biofilms, respectively. Initially, we examined the protein profiles of monospecies biofilms formed by two strains of Aggregatibacter aphrophilus, namely HK83 and CCUG 11575, along with their Hcp mutant derivatives (Hcp being a core protein for T6SS). Each strain was analyzed with six replicates (n=4 for HK83, HK83 hcp, CCUG 11575, and CCUG 11575 hcp). Subsequently, the HK83 and CCUG 11575 strains, as well as their Hcp mutant derivatives, were individually introduced into a multispecies biofilm. This multispecies biofilm consisted of seven species, namely A. actinomycetemcomitans JP2 strain (OMZ 295), Actinomyces oris (OMZ 745), Candida albicans (OMZ 110), Fusobacterium nucleatum subsp. nucleatum KP-F2 (OMZ 598), Streptococcus oralis SK248 (OMZ 607), Streptococcus mutans UA159 (OMZ 918), and Veillonella dispar ATCC 17748T (OMZ 493). These species were selected to mimic the natural co-habitat of A. aphrophilus and A. actinomycetemcomitans. Furthermore, control 7-species biofilms with A. aphrophilus strains HK83, HK83 hcp, CCUG 11575, and CCUG 11575 hcp (n=4 each) underwent proteomic analysis to gain insights into the protein expression and potential interactions within the biofilm community.

INSTRUMENT(S): Orbitrap Fusion

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

SUBMITTER: Kai Bao

LAB HEAD: Nagihan Bostanci

PROVIDER: PXD042723 | Pride | 2024-05-24

REPOSITORIES: Pride

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Bacterial symbionts in oral niche use type VI secretion nanomachinery for fitness increase against pathobionts.

Oscarsson Jan J Bao Kai K Shiratsuchi Akiko A Grossmann Jonas J Wolski Witold W Aung Kyaw Min KM Lindholm Mark M Johansson Anders A Mowsumi Ferdousi Rahman FR Wai Sun Nyunt SN Belibasakis Georgios N GN Bostanci Nagihan N

iScience 20240329 5

Microbial ecosystems experience spatial and nutrient restrictions leading to the coevolution of cooperation and competition among cohabiting species. To increase their fitness for survival, bacteria exploit machinery to antagonizing rival species upon close contact. As such, the bacterial type VI secretion system (T6SS) nanomachinery, typically expressed by pathobionts, can transport proteins directly into eukaryotic or prokaryotic cells, consequently killing cohabiting competitors. Here, we dem ...[more]

PMID: 38650989

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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: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.

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