A Bowman–Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage

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-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.

Bowman-Birk Inhibitors: Insights into Family of Multifunctional Proteins and Peptides with Potential Therapeutical Applications

“…Vigna unguiculata (Black-eyed pea) trypsin and chymotrypsin inhibitor BTCI 20 (trypsin) and 0.42 (trypsin, using surface plasmon resonance); 120 (chymotrypsin) and 0.41 (chymotrypsin, using surface plasmon resonance) [63]; 100 (proteasome T-L); 700 (proteasome ChT-L); 1400 (proteasome C-L) [60]. Crystal structure 2R33 [53], structure with trypsin 2G81 [63], structure with trypsin and chymotrypsin 3RU4 [70] Geoffroea decorticans trypsin inhibitor GdTI 2.1 (trypsin) [71]; 0.18 µM (IC 50 , α-glucosidase) [71] Apios americana trypsin inhibitor AATI 3 (trypsin); 1000 (chymotrypsin) [72] Inhibitor from Lupinus albus (White lupin) 4.2 (trypsin) [73] Luetzelburgia Auriculata ((Allemao) Ducke) Bowman-Birk inhibitor LzaBBI 0.86 (trypsin); 1.2 (chymotrypsin) [74] Inhibitors from Cajanus cajan (Red gram) 292 (trypsin); 2265 (chymotrypsin) [75] 272 (trypsin); 3725 (chymotrypsin) [76] Dolichus biflorus Bowman-Birk inhibitor 40 (trypsin); 480 (chymotrypsin) [77] Vigna mungo (Black gram) protease inhibitor BgPI 309.8 (trypsin); 10,770 (chymotrypsin) [78] Twelve Lathyrus sativus Bowman-Birk isoinhibitors Ls_BBI ranged from 6.9 to 30.8 (trypsin); ranged from 11.7 to 26.0 (chymotrypsin); Ls_BBI3c 54.6 (elastase) [79] Phaseolus acutifolius (Tepary bean) protease inhibitor TBPI 280 (trypsin); 68 (chymotrypsin) [80] Clitoria fairchildiana (Sombreiro) protease inhibitor CFPI 0.33 (trypsin); 0.15 (chymotrypsin) [81] Dioclea glabra trypsin inhibitor DgTI 0.5 (trypsin) [82] Vicia faba (Faba bean) trypsin inhibitor VFTI-G1 20.4 (trypsin) [83] Pisum sativum (Winter peas) trypsin isoinhibitors PsTI ranged from 1.2 to 0.84 (trypsin); ranged from 21 to 15 (chymotrypsin) [84]; crystal structure 1PBI [59] Rhynchosia sublobata Bowman-Birk inhibitors RsBBI 128.5 (trypsin); 807.8 (chymotrypsin) [55] Lens culinaris (Lentil) trypsin inhibitor LCTI 0.54 (trypsin); 7.25 (chymotrypsin). Solution structure 2AIH [85] Cratylia mollis (Camaratu bean) trypsin inhibitor CmTI 2 1.4 (trypsin) [86] Medicago scutellata (Snail medic) trypsin inhibitor MsTI 1.8 (trypsin) [87].…”

“…Regarding antibacterial action, BBI isolated from the seeds of leguminous plant Luetzelburgia Auriculata (LzaBBI) has exerted in vitro activity against human pathogenic Gram-positive bacteria Staphylococcus aureus with MIC and minimum bactericidal concentration (MBC) values of 23.1 × 10 −4 and 92.5 × 10 −4 µM, respectively [74]. As it is shown, LzaBBI disrupts the bacterial membrane's integrity and increases the intracellular generation of ROS, eventually leading to bacterial death.…”

Estudio de inhibidores peptídicos de proteasas aislados de alimentos de origen vegetal: potenciales aplicaciones como agentes anticoagulantes, anti-infectivos naturales y capacidad protectora del Factor de Crecimiento Epidérmico

“…Los inhibidores de proteasas de naturaleza proteica o inhibidores peptídicos de proteasas (IPPs) son polipéptidos que inhiben la acción de proteasas y se encuentran extensamente distribuidos en diferentes tejidos de animales, plantas y microorganismos (Laskowski M Jr, 1980). Muchos de ellos son pequeños péptidos que oscilan entre 15 a 60 aminoácidos, la mayoría de los cuales son hidrofóbicos y catiónicos, con un alto contenido de residuos de cisteína que forman puentes disulfuro y confieren resistencia a tratamientos térmicos, pHs extremos, fuerza iónica y proteólisis (Torres-Castillo, Jacobo, & Blanco-Labra, 2009;Macedo et al, 2011;Costa et al, 2014;Monteiro Júnior et al, 2017;Cotabarren, Tellechea, Avilés, Lorenzo Rivera, & Obregón, 2018;Martins et al, 2018).…”

“…Dado que los IPPs son moléculas pequeñas con elevada carga de residuos de cisteína, es probable que muchos de ellos presenten gran número de puentes disulfuro, lo cual les otorgaría elevada estabilidad a temperaturas y pHs extremos. Estudios realizados en los últimos años han puesto en evidencia esta premisa, reportando IPPs de plantas con destacable estabilidad fisicoquímica (Torres-Castillo et al, 2009;Macedo et al, 2011;Costa et al, 2014;Monteiro Júnior et al, 2017;Martins et al, 2018).…”

“…Otro efecto alimentario fue encontrado con inhibidores de serín proteasas que tienen la capacidad de incrementar el nivel de colecistoquinina debido a la inhibición de tripsina, por lo que pueden ser útiles para reducir el consumo de comida en humanos (Zhang, Kouzuma, Miyaji, & Yonekura, 2008 -Castillo et al, 2009;Macedo et al, 2011;H. P. S. Costa et al, 2014;Monteiro Júnior et al, 2017;Martins et al, 2018), sino porque es cada vez mayor el número de artículos científicos que reportan IPPs con actividad antimicrobiana (Satheesh & Murugan, 2011;Zhao et al, 2014;Brito et al, 2016;Habib, Fazili, Zargar, & Ganie, 2016; Maria Lígia R. Macedo et al, 2016).…”