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

Abstract

Agroinfiltration is used to treat plants with modified strains of <i>Agrobacterium tumefaciens</i> for the purpose of transient <i>in planta</i> 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 remodeling of the bacterial proteins towards enhanced infection of plant cells.

Bacterial growth‐mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming

“…Notably, outer membrane proteins play vital roles in diverse essential cellular processes, including nutrient uptake (Klebba and Newton, 1998), protein secretion and adhesion (Lauber et al ., 2018; Stubenrauch et al ., 2016), as well as membrane protein insertion and protein secretion reactions in bacteria (Baud et al ., 2014; Doyle and Bernstein, 2019). ABC transporters play an essential role in nutrient acquisition within the hostile host environment to promote bacterial survivability (Tanaka et al ., 2018), while the roles of flagellar proteins and bacterial virulence associated with surface attachment have been explored (Oberpichler et al ., 2008; Prudhomme et al ., 2021b). We also defined significant alterations of bacterial proteins by GOBP associated with motility and phosphorelay signal transduction driven by the bioreactor growth conditions (Prudhomme et al ., 2021a).…”

“…Critically, we did not assess potential confounding factors, such as timing of media component depletion and the impact of residual media components on gene expression in planta , following agroinfiltration with A. tumefaciens from bioreactor versus shake flask growth. We acknowledge that such differences may influence plant response to the bacterium; however, we anticipate a minimal impact on altered plant protein production given the dilution of the bacteria and culture media within agroinfiltration media prior to plant infection (Prudhomme et al ., 2021b). Moreover, our proteomic profiling of agroinfiltration media following inoculation with shake flask‐ or bioreactor‐grown bacteria defined clear distinction between bacterial proteomes dependent upon growth conditions, but consistency in protein abundance trends towards increased motility and virulence of bioreactor‐grown bacteria were observed.…”

“…Bacterial growth in shake flasks and bioreactors was performed as previously described (Prudhomme et al ., 2021b). From the cultures of A. tumefaciens EHA105 (pPFC0058) maintained on LB agar plates, a single colony was selected and inoculated into 5 mL of liquid LB at 28 °C and 220 rpm and cultured overnight with kanamycin (50 μg/mL) and rifampicin (25 μg/mL).…”

“…Moreover, assessment of bacterial proteome alterations during the next critical step in target protein production (i.e. agroinfiltration) revealed increased bacterial mobility and enhanced virulence‐associated protein production over a time course of exposure to agroinfiltration media (Prudhomme et al ., 2021b). These studies showcase the feasibility and value of bioreactor bacterial growth for enhanced virulence, which may influence the plant defence response, impacting production of the target protein.…”

Molecular Farming of Pembrolizumab and Nivolumab

“…To successfully agroinfiltrate expression vectors into Nicotiana benthamiana leaf cells, it is crucial to first complete an optical density (OD) measurement to ensure that the correct number of Agrobacterium tumefaciens cells are present within the agroinfiltration buffer [ 101 , 102 ]. The most frequent way to perform an OD measurement is by determining the absorbance of the cell suspension at a wavelength of 600 nm using a spectrophotometer, which allows for the rapid and precise quantification of cell number [ 102 , 103 ].…”