Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells

Abstract

bWe previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ⌬tetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its ⌬tet38, ⌬tetR21, and ⌬mgrA mutants, and a ⌬tet38 ⌬tetR21 double mutant. After 2 h of contact, the ⌬tet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ⌬tetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ⌬mgrA mutant. The growth patterns of RN6390 and the ⌬mgrA and ⌬tetR21 mutants within A549 cells were similar, while no growth was observed for the ⌬tet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes

“…1A). These data were similar to those reported in our previous study (22). After the monolayers were preincubated with anti-CD36 antibody at 50 nM, the amount of internalized wild-type RN6390 was reduced to 56% of the amount in the assay without CD36 antibody preincubation.…”

“…As we reported previously (22), Tet38 contributes to internalization of S. aureus by A549 cells. QT7 tet38 was internalized 6-fold less in A549 and human microvascular endothelial cells (HMECs) than the parent strain RN6390.…”

“…We previously demonstrated that S. aureus RN6390 internalized efficiently into the A549 lung epithelial cells (22) and that this ability was reduced in the absence of Tet38, a proton antiporter efflux pump that is capable of transporting tetracycline and certain antibacterial fatty acids (palmitoleic acid and undecanoic acid) but whose specific role in bacterial invasion and intracellular survival is not well understood (21). We thus undertook to evaluate directly the role of Tet38 in S. aureus in adherence and internalization for epithelial cells and its effect on trafficking within the host cells.…”

“…The mechanism of this effect is uncertain. Tet38 is a member of the major facilitator superfamily (MFS) which includes proton antiporters and is capable of extruding tetracycline and certain fatty acids (22). If Tet38 played a role in exchanging efflux substrates such as antibacterial fatty acids for uptake of protons, higher external proton concentrations (acidity in phagolysosome versus bacterial cell) would be expected to reduce its efflux activity and presumably also reduce its ability to protect against antibacterial fatty acids or other unknown substrates.…”

“…Tet38 is an S. aureus efflux pump that can extrude both tetracycline and unsaturated free fatty acids, such as palmitoleic acid and undecanoic acid (21,22). Tet38 plays an important role in bacterial colonization and internalization, but the mechanism of this involvement has not been explored.…”