Lantibiotics are (methyl)-lanthionine-containing antimicrobial peptides produced by several Gram-positive bacteria. Some human pathogenic bacteria express specific resistance proteins that counteract this antimicrobial activity of lantibiotics. In Streptococcus agalactiae COH1 resistance against the well-known lantibiotic nisin is conferred by, the nisin resistance protein (NSR), a two-component system (NsrRK) and a BceAB-type ATP-binding cassette (ABC) transporter (NsrFP). The present study focuses on elucidating the function of NsrFP via its heterologous expression in Lactococcus lactis. NsrFP is able to confer a 16-fold resistance against wild type nisin as determined by growth inhibition experiments and functions as a lantibiotic exporter. Several C-terminal nisin mutants indicated that NsrFP recognizes the N-terminal region of nisin. The N-terminus harbors three (methyl)-lanthionine rings, which are conserved in other lantibiotics.

Antimicrobial peptides, which contain (methyl)-lanthionine-rings are called lantibiotics. They are produced by several Gram-positive bacteria and are mainly active against these bacteria. Although these are highly potent antimicrobials, some human pathogenic bacteria express specific ABC transporters that confer resistance and counteract their antimicrobial activity. Two distinct ABC transporter families are known to be involved in this process. These are the Cpr- and Bce-type ABC transporter families, named after their involvement in cationic peptide resistance in Clostridium difficile, and bacitracin efflux in Bacillus subtilis, respectively. Both resistance systems differentiate to each other in terms of the proteins involved. Here, we summarize the current knowledge and describe the divergence as well as the common features present in both the systems to confer lantibiotic resistance.

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Clostridioides difficile is the leading cause of antibiotic-associated diarrhea but can also result in more serious, life-threatening conditions. The incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing. Against this background antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Information on the effects of AMPs on C. difficile will not only enhance the knowledge for possible biomedical application but may also provide insights into mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelope but also resulted in pronounced changes in central cellular processes such as carbohydrate metabolism. Further, the number of flagella per cell was increased during the adaptation process. The potential involvement of flagella in nisin adaptation was supported by a more resistant phenotype exhibited by a non-motile but hyper-flagellated mutant.

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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.

Treatment of bacterial infections are the great challenge of our era due to the evolved resistance mechanisms against antibiotics. The Achilles heel of bacteria is the cell wall especially during the needs of its synthesis and cell division. Here lipid II is an essential cell wall precursor component synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into the cell wall. Compounds targeting the cell wall or its biosynthesis precursors have been around for decades and have been used as antibiotics against bacterial infections like meningitis, pneumonia and endocarditis. Antimicrobial peptides (AMPs) have proven to be a promising weapon against multiresistant bacteria. However, the Bacitracin efflux (BceAB)-type ATP binding cassette transporters expressed in the membrane of human pathogenic bacteria have been shown to confer resistance to these alternative antibiotics, thereby hampering their medical development.In Streptococcus agalactiae COH1 the BceAB-type transporter NsrFP (SaNsrFP) confers highlevel resistance against the antimicrobial peptide nisin, a member of the lantibiotic subfamily. We showed that SaNsrFP provides a novel resistance mechanism by flipping lipid II back into the cytosol, thereby preventing the binding of nisin as well as other lipid II targeting compounds. This is intriguing since a relatively simple reaction mediates resistance to human pathogenic bacteria to lipid II targeting antibiotics, regardless of their structure. Significance StatementThe ABC-transporter NsrFP from Streptococcus agalactiae (SaNsrFP) belongs to the BceAB-type transporters. Several BceAB-type transporters are known to confer resistance against multiple antimicrobial peptides. In this study a new resistance mechanism was identified, which is based on the reduction of the number of cell wall precursor lipid II molecules on the cell surface mediated by SaNsrFP. SaNsrFP flips lipid II, which are considered to be the target for many antibiotics, back into the cytoplasm. With this newly gained knowledge about the resistance mechanism of BceAB-type transporters, novel strategies can be established to overcome or bypass this resistance in human pathogenic bacteria. Main Text