New insights into the resistance mechanism for the BceAB-type transporter SaNsrFP

Treatment of bacterial infections is one of the major challenges of our time due to the evolved resistance mechanisms of pathogens against antibiotics. To circumvent this problem, it is necessary to understand the mode of action of the drug and the mechanism of resistance of the pathogen. One of the most potent antibiotic targets is peptidoglycan (PGN) biosynthesis, as this is an exclusively occurring and critical feature of bacteria. Lipid II is an essential PGN precursor synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into nascent PGN. Antimicrobial peptides (AMPs), such as nisin and colistin, targeting PGN synthesis are considered promising weapons against multidrug-resistant bacteria. However, human pathogenic bacteria that were also resistant to these compounds evolved by the expression of an ATP-binding cassette transporter of the bacitracin efflux (BceAB) type localized in the membrane. In the human pathogen Streptococcus agalactiae , the BceAB transporter Sa NsrFP is known to confer resistance to the antimicrobial peptide nisin. The exact mechanism of action for Sa NsrFP is poorly understood. For a detailed characterization of the resistance mechanism, we heterologously expressed Sa NsrFP in Lactococcus lactis . We demonstrated that Sa NsrFP conferred resistance not only to nisin but also to a structurally diverse group of antimicrobial PGN-targeting compounds such as ramoplanin, lysobactin, or bacitracin/(Zn)-bacitracin. Growth experiments revealed that Sa NsrFP-producing cells exhibited normal behavior when treated with nisin and/or bacitracin, in contrast to the nonproducing cells, for which growth was significantly reduced. We further detected the accumulation of PGN precursors in the cytoplasm after treating the cells with bacitracin. This did not appear when Sa NsrFP was produced. Whole-cell proteomic protein experiments verified that the presence of Sa NsrFP in L. lactis resulted in higher production of several proteins associated with cell wall modification. These included, for example, the N -acetylmuramic acid-6-phosphate etherase MurQ and UDP-glucose 4-epimerase. Analysis of components of the cell wall of Sa NsrFP-producing cells implied that the transporter is involved in cell wall modification. Since we used an ATP-deficient mutant of the transporter as a comparison, we can show that Sa NsrFP and its inactive mutant do not show the same phenotype, albeit expressed at similar leve