Physiological stress of intracellular Shigella flexneri visualized with a metabolic sensor fused to a surface-reporter system

Guzmán, Carlos A.; Cebolla, Angel; Beltrametti, Fabricio; Staender, Lothar H.; de Lorenzo, Víctor

When deleted of its N-terminal signal-reception domain, the broad host range σ(54)-dependent transcriptional regulator XylR, along with its cognate promoter Pu, becomes a sensor of the metabolic stress of the carrier bacteria. We have employed a surface reporter system to visualize the physiological status of intracellular Shigella flexneri during infection of Henle 407 cells in culture. To this end, the xylRΔA gene has been engineered adjacent to a bicistronic transcriptional fusion of Pu to a lamB variant tagged with a short viral sequence (cor) and β-galactosidase (lacZ). The accessibility of the cor epitope to the externalmost medium and the expression of Pu in the bacterial population was confirmed, respectively, with immunomagnetic beads and the sorting of Escherichia coli cells treated with a fluorescent antibody. Intracellular Shigella cells expressed the Pu–lamB/cor–lacZ reporter at high levels, suggesting that infectious cells endure a considerable metabolic constraint during the invasion process.