TetR‐dependent gene regulation in intracellular Listeria monocytogenes demonstrates the spatiotemporal surface distribution of ActA

Abstract

To enable specific and tightly controlled gene expression both in vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic promoter Pt . Anhydrotetracycline (ATc) as inducer permitted concentration-dependent, fine-tuned expression of genes under control of the tetO operator and a suitable promoter. The actin-polymerizing ActA protein represents a major virulence factor of L. monocytogenes, required for actin-based motility and cell-to-cell spread in infected host cells. To be able to observe its spatial and temporal distribution on intracellular L. monocytogenes cells, conditional mutants featuring actA placed under TetR control were used to infect PtK2 epithelial cells. Following induction at different time intervals, the subsequent recruitment of actin by L. monocytogenes could be monitored. We found that cells displayed functional ActA after approximately 15 min, while formation of polarized actin tail was complete after 90-120 min. At this point, intracellular motility of the induced mutants was indistinguishable from wild-type bacteria. Interestingly, de novo ActA synthesis in intracellular Listeria also demonstrated the temporal, asymmetric redistribution of the membrane-anchored proteins from the lateral walls toward the cell poles.

Engineering a Dynamic Controllable Infectivity Switch in Bacteriophage T7

“…52 Phages could also be continuously propagated in a community using a "feeder" host whose susceptibility could be turned ON or OFF as needed. By tuning inducer concentrations, 53,54 phages could be engineered to repress but not eliminate specific bacterial hosts in a complex community. Alternatively, our engineered phages can act as pseudolysogenic phages, maintaining a low burden on the targeted microbe until being triggered to eliminate the host.…”

Engineering a dynamic, controllable infectivity switch in bacteriophage T7

“…Phages could also be continuously propagated in a community using a 'feeder' host whose susceptibility could be turned ON or OFF as needed. By tuning inducer concentrations 47,48 , phages could be engineered to repress but not eliminate specific bacterial hosts in a complex community. Alternatively, our engineered phages can act as pseudo-lysogenic phages, maintaining a low burden on the targeted microbe until being triggered to eliminate the host.…”

Status quo of tet regulation in bacteria

“…More recent and sophisticated modes of Tet‐ON and Tet‐OFF control are discussed later. Generic Tet‐ON regulation achieves rapid gene expression with observable phenotypic changes in as fast as 15 min, as shown in Listeria monocytogenes (Schmitter et al ., 2017 ). A return to the OFF state requires removal or dilution of the inducer.…”

“…The facultative intracellular food spoilage bacterium Listeria monocytogenes spreads from one host cell to another by means of the ActA protein. In a study by Schmitter et al (2017), the actA gene was expressed by P xyl/tet (two tetO) from the chromosome, while tetR was episomally encoded and driven by a strong synthetic promoter termed pt17 (Bertram et al, 2005). The resulting strain facilitated ATcdependent spatio-temporal control of ActA and consequently actin recruitment within epithelial human cells.…”