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Salivary MUC5B Modulation of Glucose Metabolism in Early Oral Biofilms

ABSTRACT: To investigate how early mono- and dual species biofilms of oral Actinomyces naeslundii and Streptococcus gordonii clinical isolates metabolized glucose in the presence of the complex salivary mucin MUC5B, this study employed NMR-based metabolomics with interpretation of network integration. Untargeted NMR data analysis for metabolite detection and annotation and in silico studies of biochemical relationships between metabolites and cellular processes was performed to study functional differences in the early biofilm metabolomes. Significant differences in metabolite abundance elicited by the presence of MUC5B were detected. The main metabolites that distinguished the biofilms grown with 25% MUC5B with 20 mM glucose (MUC5B+G) from those grown with physiologic buffer with 20 mM glucose (PBS+G) were the unannotated metabolites 68 and 73. In the conditions were they were each present, pyruvate, ethanol and metabolite 134 were significantly more abundant in the presence of MUC5B, while metabolites 84, 97 and sarcosine were significantly less abundant. The pathway enrichment analysis of the metabolites that were differently expressed in early A. naeslundii, S. gordonii and dual species biofilms grown with PBS+G or MUC5B+G showed that pyruvate metabolism was significantly over-represented. Studying the metabolic interactions between commensal members of oral biofilms and modulatory effects of host factors such as glycoproteins in saliva during metabolism of substrates that are potential drivers of dysbiosis, such as glucose, is essential to understand the roles of oral microbial ecosystems in oral health and disease.

INSTRUMENT(S): Nuclear Magnetic Resonance (NMR) -

PROVIDER: MTBLS10078 | MetaboLights | 2024-09-11

REPOSITORIES: MetaboLights

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Project description:A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in M-bM-^HM-^FmifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and M-NM-^TmifR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturerM-bM-^@M-^Ys instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326. Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.

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Salivary MUC5B Modulation of Glucose Metabolism in Early Oral Biofilms

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