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

Abstract

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 SaNsrFP is known to confer resistance to the antimicrobial peptide nisin. The exact mechanism of action for SaNsrFP is poorly understood. For a detailed characterization of the resistance mechanism, we heterologously expressed SaNsrFP in Lactococcus lactis. We demonstrated that SaNsrFP 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 SaNsrFP-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 SaNsrFP was produced. Whole-cell proteomic protein experiments verified that the presence of SaNsrFP 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 SaNsrFP-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 SaNsrFP and its inactive mutant do not show the same phenotype, albeit expressed at similar levels, which demonstrates the ATP dependency of the mediated resistance processes. Taken together, our data agree to a target protection mechanism and imply a direct involvement of SaNsrFP in resistance by shielding the membrane-localized target of these antimicrobial peptides, resulting in modification of the cell wall.

After a century of nisin research - where are we now?

“…The latest studies regarding NsrFP has provided fresh insights into the mechanism of BceAB-type transporters (Gottstein et al 2022 ). An unusual feature of these systems is that both transporter permease and the histidine kinase component of the TCS are thought to be mutually indispensable for both sensing of and resistance to the antimicrobial, forming a sensory complex in which the transporter represents the actual sensor (Dintner et al 2011 , Clemens et al 2018 ).…”

“…However, heterologous expression of NsrFP alone (i.e. without its cognate TCS) in L. lactis conferred resistance to nisin (Reiners et al 2017b ) and bacitracin, as well as a number of other lipid II targeting compounds (Gottstein et al 2022 ). Moreover, comparative proteomic analysis of L. lactis cells expressing NsrFP with a nonfunctional mutant (NsrF H202A P) suggests that NsrFP may also act by shielding lipid II cycle intermediates from the antimicrobial compound (Gottstein et al 2022 ), and thereby provide additional resistance through target protection of cell wall synthesis in agreement with previous studies on BceAB-like systems (Kobras et al 2020 ).…”