A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage Thiago F. Martins, Ilka M. Vasconcelos, Rodolpho G. G. Silva, Fredy D. A. Silva, Pedro F. N. Souza, Anna L. N. Varela, Louise M. Albuquerque, and Jose T. A. Oliveira* Laboratory of Plant Defense, Department of Biochemistry and Molecular Biology, Federal University of Ceara (UFC), Avenida Mister Hull, 60451-970, Fortaleza, Ceara, Brazil ABSTRACT: Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor (LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH 2 -terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus. O wing to increasing medical awareness of the rapid emergence of multidrug-resistant bacteria worldwide, endangering the efficacy of antibiotics and threatening the healthcare systems, two articles were recently published on the antibiotic resistance crisis. 1,2 In part 1, in which the causes and threats are discussed, the antibiotic resistance crisis has been attributed to the overuse, inappropriate prescription, extensive agricultural use as growth supplements in livestock, and the availability of only a few novel antibiotics due to economic and regulatory obstacles. 1 In part 2, which advanced management strategies and new antibiotics, the author discussed steps to reduce antibiotic resistance by improving diagnosis, tracking, and prescribing correct practices; optimizing therapeutic regimens; eliminating diagnostic uncertainty; and improving tracking methodologies. These procedures should be used by healthcare facilities to electronically report infections, anti- biotic use, and resistance, besides preventing infection transmission, which can significantly decrease resistance by eliminating the need for antibiotics. 2 Staphylococcus aureus and its methicillin-resistant strains (MRSA) are Gram-positive bacteria that exist in community and hospital settings that often cause bloodstream infections in hospitalized patients. Hospital-acquired MRSA (HA-MRSA) strains are generally multidrug resistant, and although vancomycin has been used to treat MRSA infections, a few cases of vancomycin-resistant S. aureus (VRSA) have recently emerged. 3,4 In connection with this recognized reality of rapid global emergence of antibiotic-resistant bacteria, new strategies of treatments are needed, either by chemical modification of existing drugs or purification of biomolecules with therapeutic potential. 5,6 Several proteins with deleterious actions against bacteria have been isolated from plants. Protease inhibitors (PIs) are proteins or peptides that complex with proteolytic enzymes, inhibiting their catalytic activity. 7 PIs are ubiquitous in nature, and in plants the highest concentrations are generally found in seeds. PIs perform a variety of functions during plant development, as they control protein degradation during seed dormancy and act as plant defense molecules against insect and pathogen attacks. 8-10 Over the last years, PIs have received greater attention due to the possibility of various applications in both human health and crop protection. 11,12 Indeed, recent studies reported that some PIs of plant origin like those from Abelmoschus moschatus (AMTI-II) 13 and Jatropha curcas (JcTI-I) 14 kill human pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Bacillus subtilis, Streptococcus pneumoniae, Bacillus cereus, and Salmonella enteric) by damaging the plasma cell membrane. 8 They also have antifungal, antiprotozoal, and antiviral activities. 9 Others, like those from Clitoria fairchildiana (CFPI), 15 Ricinus communis (RcTI), 16 and Cassia leiandra (ClTI), 17 inhibit Received: June 26, 2017 Article pubs.acs.org/jnp Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.7b00545 J. Nat. Prod. XXXX, XXX, XXX-XXX Downloaded via UNIV OF TOLEDO on June 22, 2018 at 03:50:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.