Method: Carious lesions from 3 children were sampled. Each sample was inoculated into paired CDC biofilm reactors containing basal mucin medium, and microcosms were grown per Rudney et al. J. Appl. Microbiol. 115:1340, 2012. One reactor was sucrose-pulsed 5 times daily (WS); the other was not (NS). DNA extracts were sent for HOMIM 16s rDNA analysis. Biofilm proteins were extracted using pressure cycling, and tryptic digests were analyzed by 1-D MSMS, using an Orbitrap Velos mass spectrometer. Oral microbial peptides were identified in the Galaxy-P platform, using a two-step workflow (Jagtap et al. Proteomics 12:992, 2012). MEGAN4 software was used to compare normalized spectral counts.
Result: Each of the NS microcosms showed individual differences in protein expression patterns at the genus and pathway levels. The 3 WS microcosms were more similar to each other, and very different from their NS counterparts. Reactor pH dropped below 5.5 during sucrose pulsing. The relative abundance of Streptococcus 16S rDNA and protein spectral counts increased, while protein expression by other genera was suppressed. Interestingly, Streptococcus mutanswas detected only in one WS microcosm, at very low levels. The expression of glycolysis proteins, lactate dehydrogenase, ammonia assimilation proteins, and acid tolerance proteins increased. By contrast, arginine deiminase pathway proteins and pyruvate formate lyase decreased.
Conclusion: The oral microcosm approach facilitates metaproteomic analysis of changes induced by frequent sucrose exposure, making possible deep exploration of biofilm community changes in protein expression induced by dysbiosis.