Lung inflammatory lesions caused by <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> infection were reduced by Polydim-I peptide treatment.

<p>Twenty-six days after the challenge, the lungs were processed, sectioned, HE stained, and examined at 40 × or 100 × magnification. (A and B) normal lungs from PBS treated control group at 40 × and 100 × magnifications, respectively; inflammatory lesions observed in the lungs of the infected group 40 × (C) and 100 × (D). Lungs from animals infected and treated with Polydim-I (2 mg/kg/mLW) 40 × (E) and 100 × (F). The area of the inflammatory lesions were calculated and plotted in a graph (G). One-way ANOVA followed by Dunn’s test was used to determine significant differences (* p<0.05).</p

<i>M</i>. <i>abscessus subsp</i>. <i>massiliense</i> bacillary load reduction in IFN-γKO-infected mice after treatment with Polydim-I peptide.

<p>IFN-γKO mice were infected intravenously with 10⁶ CFU of <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i>. Eighteen days after infection, the mice were treated with Polydim-I peptide at three different dosages (2, 1 or 0.5 mg/kg/mLW) or with clarithromycin (CLR 200 mg/kg/mLW). As a control group, infected mice were treated with PBS. Percentages of bacterial load reduction in the lungs (A), spleen (B) and liver (C) from each treatment group were determined relative to the control PBS group (* p<0.05).</p

Morphology of <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> cell surface upon the action of Polydim-I peptide.

<p>(A) Scanning Electron microscopy of untreated <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> cells. (B) <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> exposed to 3.8 μg/mL of Polydim-I peptide for 24 hours, presenting mild surface alterations (white arrows). <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> with severe cell wall disruptions after treatment with 7.6 μg/mL of Polydim-I peptide for 24 hours (C). (D) <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> treated with clarithromycin (0.5 μg/mL) without visible cell wall damage. (A) and (C) 10,000 × magnification. (B) and (D) 12,000 × magnification.</p

Antimycobacterial Activity of a New Peptide Polydim-I Isolated from Neotropical Social Wasp <i>Polybia dimorpha</i>

<div><p><i>Mycobacterium abscessus</i> subsp. <i>massiliense</i>, a rapidly growing mycobacteria (RGM) that is becoming increasingly important among human infectious diseases, is virulent and pathogenic and presents intrinsic resistance to several antimicrobial drugs that might hamper their elimination. Therefore, the identification of new drugs to improve the current treatment or lower the risk of inducing resistance is urgently needed. Wasp venom primarily comprises peptides that are responsible for most of the biological activities in this poison. Here, a novel peptide Polydim-I, from <i>Polybia dimorpha</i> Neotropical wasp, was explored as an antimycobacterial agent. Polydim-I provoked cell wall disruption and exhibited non-cytotoxicity towards mammalian cells. Polydim-I treatment of macrophages infected with different <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> strains reduced 40 to 50% of the bacterial load. Additionally, the Polydim-I treatment of highly susceptible mice intravenously infected with <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> induced 0.8 to 1 log reduction of the bacterial load in the lungs, spleen, and liver. In conclusion, this is the first study to show the therapeutic potential of a peptide derived from wasp venom in treating mycobacteria infections. Polydim-I acts on the <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> cell wall and reduce 40–90% of the bacterial load both <i>in vitro</i> and <i>in vivo</i>. The presented results encourage further studies on the use of Polydim-I as one of the components for <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> treatment.</p></div

<i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> CFU in infected macrophages treated with Polydim-I peptide.

<p>Peritoneal macrophages from BALB/c mice were infected with 6 isolates of <i>M</i>. <i>abscessus</i> subsp. <i>massiliense</i> (A thru F) or the reference strains CRM0020 (G) and ATCC19977 (H) at a MOI of 10:1, and treated with 7.6 μg/mL of Polydim-I peptide or with 1 μg/mL of clarithromycin. Bacterial load of phagocytosed bacteria were determined at three hours after infection and total recovered bacteria from supernatant and macrophages at 72 hours after infection were determined for non-treated (closed circles), peptide treated (closed squares), and clarithromycin treated (open circles). The data represent mean of quadruplicates and is representative of three independent experiments. One-way ANOVA followed by Dunn’s test was used to determine significant differences (* p<0.05).</p

Percentage of mycobacterial load reduction in infected macrophages by Polydim-I treatment.

<p>The CFU in peritoneal macrophages, 72 hours after treatment with Polydim-I (white bars) or clarithromycin (black bars), was determined and the percentage of inhibition was calculated in relation to non-treated macrophage cultures. The data represent mean of quadruplicates and is representative of three independent experiments. One-way ANOVA followed by Dunn’s test was used to determine significant differences (* p<0.05).</p

Chromatographic profile, mass spectra and <i>de novo</i> sequencing of Polydim-I peptide.

<p>(A). Chromatographic profile obtained after separation of the low molecular weight compounds from <i>Polybia dimorpha</i> venom through reversed phase high performance liquid chromatography. The fraction containing the peptide Polydim-I is signalized. (B) Mass spectra of fraction 12 obtained through MALDI TOF/TOF, with the major ion m/z 2441.7 [M+H]+. (C). <i>De novo</i> sequencing of Polydim-I using MS/MS. The ion series y is written above with the 22 amino acids represented by letters.</p

J774 macrophage cell line cytotoxic effects and hemolytic activity of Polydim-I peptide.

<p>J774 macrophages were exposed to different concentrations of Polydim-I peptide (7.6, 15.2, 60.8, or 121.6 μg/mL) for 3 days. Cell viability percentage was determined by LDH release assay (A). In (B), the percentage of human red blood cells lysis. The results represent three independent experiments.</p