Quantitative proteomics insights into Chlamydomonas reinhardtii thermal tolerance enhancement by a mutualistic interaction with Sinorhizobium meliloti.

Interactions between photosynthetic microalgae and bacteria impact the physiology of both partners, which influence the fitness and ecological trajectories of each partner in an environmental context-dependent manner. Thermal tolerance of Chlamydomonas reinhardtii can be enhanced through a mutualistic interaction with vitamin B12 (cobalamin)-producing Sinorhizobium meliloti. Here, we used label-free quantitative proteomics to reveal the metabolic networks altered by the interaction under normal and high temperatures. We created a scenario where the growth of Sinorhizobium requires carbon provided by Chlamydomonas for growth in co-cultures, and survival of Chlamydomonas under high temperatures relies on cobalamin and possibly other metabolites produced by Sinorhizobium. Differential abundance analysis identified proteins produced by each partner in co-cultures compared to mono-cultures at each temperature. Proteins involved in cobalamin production by Sinorhizobium increased in the presence of Chlamydomonas under elevated temperatures, whereas in Chlamydomonas, there was an increase in cobalamin-dependent methionine synthase and certain proteins associated with methylation reactions. Co-cultivation and heat stress strongly modulated the central metabolism of both partners as well as various transporters that could facilitate nutrient cross-utilization. Co-cultivation modulated expression of various components of two- or one-component signal transduction systems, transcriptional activators/regulators, or sigma factors, suggesting complex regulatory networks modulate the interaction in a temperature-dependent manner. Notably, heat and general stress-response and antioxidant proteins were upregulated in co-cultures, suggesting that the interaction is inherently stressful to each partner despite the benefits of mutualism. Our results shed insight into the metabolic tradeoffs required for mutualism and how metabolic networks are modulated by elevated temperature. IMPORTANCE: Photosynthetic microalgae are key primary producers in aquatic ecosystems, playing an important role in the global carbon cycle. Nearly every alga lives in association with a diverse community of microorganisms that influence each other and their metabolic activities or survival. One chemical produced by bacteria that influence algae is vitamin B12, an enzyme cofactor used for a variety of metabolic functions. The alga Chlamydomonas reinhardtii benefits from vitamin B12 produced by Sinorhizobium meliloti by producing the amino acid methionine under high temperatures which are required for Chlamydomonas thermotolerance. Yet, our understanding of this interaction under normal and stressful temperatures is poor. Here, we used quantitative proteomics to identify differentially expressed proteins to reveal metabolic adjustments made by Chlamydomonas and Sinorhizobium that could facilitate this mutualism. These findings will enhance our understanding of how photosynthetic algae and their associated microbiomes will respond as global temperatures increase.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.