Multidrug-resistant Staphylococcus aureus infections pose a significant threat to human health. Antibiotic resistance is most commonly propagated by conjugative plasmids like pLW1043, the first vancomycin-resistant S. aureus vector identified in humans. We present the molecular basis for resistance transmission by the nicking enzyme in S. aureus (NES), which is essential for conjugative transfer. NES initiates and terminates the transfer of plasmids that variously confer resistance to a range of drugs, including vancomycin, gentamicin, and mupirocin. The NES N-terminal relaxase-DNA complex crystal structure reveals unique protein-DNA contacts essential in vitro and for conjugation in S. aureus. Using this structural information, we designed a DNA minor groove-targeted polyamide that inhibits NES with low micromolar efficacy. The crystal structure of the 341-residue C-terminal region outlines a unique architecture; in vitro and cell-based studies further establish that it is essential for conjugation and regulates the activity of the N-terminal relaxase. This conclusion is supported by a smallangle X-ray scattering structure of a full-length, 665-residue NES-DNA complex. Together, these data reveal the structural basis for antibiotic multiresistance acquisition by S. aureus and suggest novel strategies for therapeutic intervention.A ntibiotic resistance, which arises in bacterial pathogens through conjugative plasmid DNA transfer, is a well-established threat to global health. For example, whereas vancomycin has been essential in treating recalcitrant Staphylococcus aureus infections for decades, vancomycin-resistant S. aureus (VRSA) strains have now appeared in clinical settings worldwide (1-3). VRSA first arose in the United States through the interplay of conjugative DNA transfer and resistance-determinant transposition. The resulting plasmid, pLW1043, has been sequenced and contains not only a vanHAX vancomycin-resistance transposon, but also a cadre of putative DNA transfer genes (4). It was recently shown that the S. aureus plasmid pSK41, which is closely related to pLW1043, mediates the transfer of vancomycin resistance from Enterococcus faecalis into strains of methicillin resistant S. aureus (MRSA) (5). Conjugative bacterial plasmids use almost exclusively plasmid-encoded factors that work in concert to coordinate the cell-to-cell transfer of one strand of the duplex plasmid (6, 7). An element common to all conjugative processes is the plasmid-encoded relaxase enzyme that initiates and terminates transfer by creating a transient single-strand DNA break and covalent protein-DNA intermediate (8,9).The vancomycin-resistance plasmid pLW1043 (4) and related plasmids from S. aureus (10, 11), including pSK41 and pGO1 (12-14) as well as plasmids from streptococcal, lactococcal, and clostridial strains, encode a relaxase enzyme termed nicking enzyme in Staphylococcus (NES) that exhibits a unique fulllength sequence (Fig. S1). It is 665 residues in length, confines its relaxase motifs to its N-terminal ∼220 aa, an...

Asthma is a chronic airway disease characterized by inflammation, mucus hypersecretion and abnormal airway smooth muscle (ASM) contraction. Bacterial permeability family member A1, BPIFA1, is a secreted innate defence protein. Here we show that BPIFA1 levels are reduced in sputum samples from asthmatic patients and that BPIFA1 is secreted basolaterally from healthy, but not asthmatic human bronchial epithelial cultures (HBECs), where it suppresses ASM contractility by binding to and inhibiting the Ca 2 þ influx channel Orai1. We have localized this effect to a specific, C-terminal a-helical region of BPIFA1. Furthermore, tracheas from Bpifa1 À / À mice are hypercontractile, and this phenotype is reversed by the addition of recombinant BPIFA1. Our data suggest that BPIFA1 deficiency in asthmatic airways promotes Orai1 hyperactivity, increased ASM contraction and airway hyperresponsiveness. Strategies that target Orai1 or the BPIFA1 deficiency in asthma may lead to novel therapies to treat this disease.

These data identify a critical role for mast cells in the development of many features of a mouse model of eosinophilic CRS, suggesting that therapeutic strategies targeting mast cells be examined in humans afflicted with this disease.