Evaluation of an Antimicrobial L-Amino Acid Oxidase and Peptide Derivatives from Bothropoides mattogrosensis Pitviper Venom

Healthcare-associated infections (HAIs) are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO) is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO) was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3) were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms

Cytotoxic activities of Bm-LAO, fractions 7, 44, 51, 68 the fragments 1, 2 and 3 against RAW264.7 cells and human erythrocytes.

<p>Samples (512 µg.mL⁻¹) were incubated with the cells for 24 h. Cell viability was assessed by MTT assay and hemolytic assay. Values are expressed mean ± S.D. for three independent experiments.</p>*<p>p<0.05, compared to negative control (PBS buffer, pH 7.4). CVBm: <i>B. mattogrosensis</i> crude venom; Bm-LAO: <i>B. mattogrosensis</i> L-amino acid oxidase.</p

Enzyme activity of <i>B. mattogrosensis</i> L-amino acid oxidase (Bm-LAO).

<p>Values are expressed mean ± S.D. for three independent experiments.</p>*<p>p<0.05, compared to negative control (PBS buffer, pH 7.4);</p>**<p>p<0.01 compared to negative control.</p

Antibacterial activity of CVBm, fractions obtained by gel filtration, HPLC chromatography and fragments obtained after <i>de novo</i> sequencing.

*<p>MIC (Minimum Inhibitory Concentration) is defined as the lowest concentration that inhibited 100% of bacterial growth.</p><p>Inhibition by each sample was determined after 12 h incubation. <b>CV</b><b><i>Bm</i></b>: <i>B. mattogrosensis</i> crude venom; <b><i>Bm</i></b><b>LAO</b>: <i>B. mattogrosensis</i> L-amino acid oxidase; <b><i>Bm</i></b><b>LAO-f1, -f2 and -f3</b>: <i>B. mattogrosensis</i> L-amino acid oxidase fragments 1, 2 and 3.</p

Multiple alignments of protein fragments determined by MALDI ToF/ToF of BmLAO against a wide diversity of L-amino oxidases from different snake species.

<p>The black coils represent α-helices and black arrows β-sheets. Numbers represent the positions of amino acid residues obtained from <i>Gloydius halys</i> (pdb 1tdk) L-amino-oxidase.</p

Theoretical three-dimensional structures of peptide derivatives from <i>Vipera ammodytes ammodytes</i> L-amino-oxidase (A) BmLAO-f1, (B) BmLAO-f2 and (C) BmLAO-f3.

<p>Exposed residues probably involved in membrane interaction are labelled and side chains are also represented.</p

Three-dimensional structure of L-amino-oxidase from <i>Vipera ammodytes ammodytes</i> (pdb 3kve) and theoretical models of peptide derivatives (A) BmLAO-f1, (B) BmLAO-f2 and (C) BmLAO-f3.

<p>Blue and red regions correspond to the cationic and anionic areas, respectively. The structure was visualized using PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>).</p