Quantitative proteomic profiling of shake flask versus bioreactor growth reveals distinct responses of Agrobacterium tumefaciens for preparation in molecular pharming.

The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Comparative study of the effects of optic design on lens epithelium in vitro.

We performed two tissue culture experiments designed to compare the effects of various posterior chamber optics on lens epithelium. In the first experiment, we recorded, by phase contrast microphotography, the migration of rabbit lens epithelium exoplants placed adjacent to the optic of various posterior chamber lenses. In the second experiment, we used phase contrast microphotographs to document the effects of various posterior chamber optics when gently placed on a confluent layer of rabbit lens epithelium. From our in vitro studies, we conclude the following: There is inhibition of lens epithelial migration and even cytotoxic effects from direct contact with polymethylmethacrylate optics; glass optics have appreciably less effect on lens epithelium; polymethylmethacrylate optics with ridges (complete annulus or incomplete) do not inhibit lens epithelial migration as well as planoconvex lenses, and they do not have a cytotoxic effect except at the points of contact between the ridge and the supporting surface.

Lens epithelial inhibition by PMMA optic: implications for lens design.

It has been a clinical impression that posterior chamber lens implants in some way inhibit opacification of the posterior lens capsule after extracapsular cataract extraction. The mechanism of this inhibition is unclear; it may be related to mechanical contact or blockage of migration of lens epithelial cells, or possibly to the leeching of toxic factors from the lens itself. A better understanding of the exact mechanism of opacification inhibition may have important clinical implications for intraocular lens design. For example, some lens designs that facilitate Nd:YAG capsulotomy by physically separating the posterior chamber lens and the posterior capsule may result in less inhibition and in fact more opacification of posterior capsules. We performed in vitro tissue culture studies of the effect of the polymethylmethacrylate (PMMA) optic of a planoconvex intraocular lens on cultured rabbit lens epithelium. These studies demonstrated both inhibition of lens epithelial migration beneath the PMMA optic (plano side down) as well as metaplasia and necrosis of lens cells growing directly beneath the optic. The clinical implications of these studies for intraocular lens design are discussed.