Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.

Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.

Beta-catenin is known to be a vital component of the canonical Wnt signaling cascade, involved in the carcinogenesis of different solid tumors. We aimed to evaluate the effects of Beta-catenin inhibition in head and neck squamous cell carcinoma (HNSCC) in vitro. The small molecular compound MSAB was used to inhibit Wnt/Beta-catenin signaling in a human papillomavirus (HPV)-positive and HPV-negative cell line and its effects on cell proliferation, migration, colony formation, apoptosis, as well as radiosensitizing properties were assessed. Significant antineoplastic effects were observed in both cell lines. Interestingly, stronger anti-neoplastic and radiosensitizing effects were observed in the HPV-negative cell line, whereas stronger anti-migratory potential was detected in HPV-positive HNSCC cells. In conclusion, our findings suggest MSAB as a potential therapeutic agent for HNSCC. Further studies are warranted to unravel the mechanistic background of our findings.

New chemotherapy agents are warranted for head and neck squamous cell carcinoma (HNSCC), particularly for incidence-rising HPV-positive tumors. Based on the evidence of Notch pathway involvement in cancer promotion and progression, we aimed to gain insights into the in vitro antineoplastic effects of gamma-secretase inhibition in HPV-positive and -negative HNSCC models.