Antimicrobial and Chemotactic Activity of Scorpion-Derived Peptide, ToAP2, against Mycobacterium massiliensis

Abstract

Mycobacterium massiliense is a rapid growing, multidrug-resistant, non-tuberculous mycobacteria that is responsible for a wide spectrum of skin and soft tissue infections, as well as other organs, such as the lungs. Antimicrobial peptides had been described as broad-spectrum antimicrobial, chemotactic, and immunomodulator molecules. In this study we evaluated an antimicrobial peptide derived from scorpion Tityus obscurus as an anti-mycobacterial agent in vitro and in vivo. Bioinformatics analyses demonstrated that the peptide ToAP2 have a conserved region similar to several membrane proteins, as well as mouse cathelicidin. ToAP2 inhibited the growth of four M. massiliense strains (GO01, GO06, GO08, and CRM0020) at a minimal bactericidal concentration (MBC) of 200 µM. MBC concentration used to treat infected macrophages was able to inhibit 50% of the bacterial growth of all strains. ToAP2 treatment of infected mice with bacilli reduced the bacterial load in the liver, lung, and spleen, similarly to clarithromycin levels (90%). ToAP2 alone recruited monocytes (F4/80^low Gr1), neutrophils (F4/80^- Gr1), and eosinophils (F4/80+ Gr1+). ToAP2, together with M. massiliense infection, was able to increase F4/80^low and reduce the percentage of F4/80^high macrophages when compared with infected and untreated mice. ToAP2 has in vitro anti-microbial activity that is improved in vivo due to chemotactic activity.

Keywords: Mycobacterium; antimicrobial peptide; monocyte; mycobacteria inhibition; neutrophil.

Figure 1

In silico analyses of ToAP2 and its conserved residues. (A) Pairwise alignment of ToAP2 and the proteins Granulysin, NK-Lysin, CRAMP, and LL-37, provided by the LALIGN online server. (B) Predicted secondary structure was provided by the PSIPRED tool, generated under sequence homology with 10 other sequences. (C) Multiple sequence alignment of ToAP2 with a conserved portion of membrane proteins, were provided by Clustal Omega. (D) Structural alignment with human smoothened 7TM protein, was generated by Chimera software and Emboss Needle.

Figure 2

Growth inhibition of isolates and reference Mycmas strain by the scorpion peptide ToAP2. Peptide ToAP2 was used from 6.5 to 200 μM concentrations, against three clinical isolates (GO01 (A), GO06 (B), and GO08 (C)) and a reference strain (CRM0020 (D)). The percent inhibition was determined by comparison with control without peptide or any other antimicrobial. The MBC concentration was determined, for each strain, as the concentration that had no CFU after peptide treatment. CLR: clarithromycin.

Figure 3

ToAP2 reduction of bacillary load in macrophages infected with Mycmas. Bone marrow-derived macrophages were infected with Mycmas strains (MOI 1:10) and then treated with ToAP2 MBC concentrations. Graph (A) represent GO01 strain, (B) GO06, (C) GO08, and (D) CRM0020. Graph (E) represents the percentage inhibition of bacterial load in all experiments and treatments. CFU: colony forming units. Differences between the means of the groups were determined by ANOVA and significant differences with p < 0.05 are shown with an asterisk (*), n = 4.

Figure 4

ToAP2 inhibits the bacillary load of Mycmas in organs of infected mice. IFN-γ KO mice were infected with Mycmas and treated with three different concentrations of ToAP2 (0.5 mg/kg, 1 mg/kg, and 2 mg/kg). After the treatment, the lung (A), liver (B), and spleen (C) were collected and the CFUs in these organs were evaluated. (D) shows the percentage inhibition of bacterial load in all organs and treatments. Differences between the means of the groups were determined by ANOVA and significant differences with the control, with p < 0.05 are shown with an asterisk (*). Significant differences between the other groups with p < 0.05 are shown with a hash symbol (#), n = 4.

Figure 5

Chemotactic activity of ToAP2 in BALB/c mice peritoneum. Four animals from each group were injected intraperitoneally with 2 mg/kg of the peptide ToAP2 (in 100 μL) or with 100 μL of PBS. Twenty-four hours after the inoculation, mice were euthanized and the cells present in the peritoneum were evaluated. Differences between the means of the groups were determined by Student’s t-test and significant differences with the control, with p < 0.05, are shown with an asterisk (*), n = 4. Top figures show the dot plots of the acquired cells comparing GR-1 PerCP-Cy5.5 versus F4/80 APC. (A): Percentage of large peritoneal macrophages (F4/80^high), (B): Percentage of small peritoneal macrophages (F4/80^low), (C): Percentage of neutrophils (Ly6G+), (D): Percentage of eosinophils (Ly6G⁺F4/80⁺).