TonB-Dependent Transport of Thiopeptide Antibiotics to Kill Gram-Negative Pathogens

Abstract

The outer membrane (OM) of P. aeruginosa is a semi-permeable barrier that contributes to antibiotic resistance by reducing uptake. Finding strategies to circumvent this barrier is a major challenge. One approach involves screening in physiologically relevant conditions to identify novel activity in existing molecules. We discovered that thiostrepton (TS), a thiopeptide antibiotic with no reported activity against Gram-negative bacteria, hijacks the pyoverdine siderophore transporters FpvA and FpvB to cross the OM under iron limitation to inhibit translation. Using TS, we subsequently showed that FpvB is not primarily a pyoverdine transporter, but rather a promiscuous transporter for siderophores ferrichrome and ferrioxamine B. Our work with TS suggested that other thiopeptides may use siderophore transporters for entry into the cell. This hypothesis led to a screen to identify other thiopeptides with activity against P. aeruginosa, uncovering two other thiopeptides, thiocillin and micrococcin, that use the ferrioxamine transporter FoxA for uptake. We discovered another siderophore, bisucaberin, could also use FoxA for uptake and our collaborators solved the crystal structure of bisucaberin bound to FoxA. Through biochemical approaches, we characterized how FoxA accommodates structurally distinct ligands. Finally, we screened known large natural product antibiotics with no pseudomonal activity under nutrient limitation and discovered that the glycopeptide vancomycin inhibits growth by blocking peptidoglycan crosslinking. This pilot screen emphasizes the importance of screening for antibiotics under physiologically relevant conditions to avoid overlooking potential hits. Overall, the findings from these studies can be used to guide medicinal chemistry efforts to develop novel siderophore-antibiotic conjugates for the treatment of P. aeruginosa infections. These results also help us gain a deeper understanding of the mechanism of binding and uptake through siderophore transporters and the range of substrates that can be taken up.