Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin

Abstract

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.

Keywords: animal experimentation; drug targets; mass spectrometry; modeling; peptides as drugs; side effects.

Figure 1

The antimicrobial peptide 6027 inhibits Toxoplasma gondii at nanomolar concentrations. The inhibition of T. gondii RH tachyzoite proliferation was determined using a beta-galactosidase reporter strain in the presence of a concentration series of peptide 6027 added prior to the infection of confluent HFF by tachyzoites. The impact of peptide 6027 on the vitality of uninfected HFF was assessed via resazurin reduction. Mean values ± SE are indicated for quadruplicates. The x-axis scale is logarithmic at basis 10.

Figure 2

Ultrastructure of T. gondii tachyzoites cultured in human foreskin fibroblasts (HFF). Parasites proliferated intracellularly within a parasitophorous vacuole (A,C), and corresponding higher magnification views are depicted in (B) and (D–E). A tachyzoite undergoing endodyogeny forming a daughter zoite (dz) is shown in (F), and structural details of the mitochondrion are shown at higher magnification in (G). Notice distinct apicomplexan organelles at the anterior end of tachyzoites such as the conoid (con), rhoptries (rop) and micronemes (mic). Dense granules (dg) and parts of the mitochondrion (mt) are dispersed within the cytoplasm; nuc = nucleus. Bars in (A) = 1 µm; (B) = 0.35 µm; (C) = 1 µm; (D) = 0.55 µm; (E) = 0.42 µm; (F) = 0.55 µm; (G) = 0.18 µm.

Figure 3

Ultrastructure of T. gondii tachyzoites grown in HFF exposed to peptide 6027 during 6 h (A–D), 24 h (E) and 48 h (F). (A,C) are lower-magnification views, while (B,D) show more detailed views at higher magnification. The bold arrow in A points to a tachyzoite located in a vacuole that is filled with an electron-dense matrix (ma), and the thin arrows in (F) indicate the lack of a defined parasitophorous vacuole membrane (pvm) after 48 h of treatment (F); ant = anterior end; dz = emerging daughter zoites; dg = dense granules; hnuc = host nucleus; rop = rhoptries; mic = micronemes; nuc = nucleus; con = conoid; v = vacuolization; mt = mitochondrial residues; ld = lipid droplets. Bars in (A) = 1.1 µm; (B) = 0.35 µm; (C)= 0.75 µm; (D) = 0.28 µm; (E,F) = 0.75 µm.

Figure 4

Cerebral parasite load (A) and IgG-titers (B) in CD1 mice infected with T. gondii oocysts and either treated with peptide 6027 (3 mg/kg/day for 5 days) or treated with placebo only (C+). C− was not infected and only placebo treated; * indicates p < 0.05; ns = not significant.

Figure 5

IFN-γ responses of splenocyte cultures in CD1 mice infected with T. gondii oocysts and either treated with peptide 6027 (3 mg/kg/day for 5 days) or treated with placebo only (C+). C− was not infected and only placebo treated; ns indicates no statistically significant difference; * = p < 0.05; *** indicates p = 0.0001; ns, not significant.

Figure 6

Effects of peptide 6027 on murine T cells (A) and B cells (B) with respect to viability and proliferation in vitro; 96-well plates were seeded with splenocytes obtained from murine spleen (2 × 10⁶ cells/mL, 100 µL/well), and were exposed to ConA (5 µg/mL) or LPS (10 µg/mL). Peptide 6027 was added at 0.1, 0.5, 1 and 2 µM, respectively, and cultivation was carried out for 48 h at 37 °C/5% CO₂. Viability was assessed by resazurin reduction and is given as relative fluorescence units (RFU); the proliferation of cells was measured by BrdU ELISA and is given as absorption at 450 nm (A₄₅₀).

Figure 7

Principal component analysis of affino-proteome data sets from T. gondii RH tachyzoites (A,B) and from Mus musculus spleens (C,D). Cell-free extracts were prepared and subjected to differential affinity chromatography on mock (red square), peptide 21,358 (blue triangle) or peptide 6027 (green circle) columns followed by mass spectrometry as described in the Materials and Methods. (A,C), Top3 data; (B,D), LFQ data. X-axis, principal component 1; Y-axis, principal component 2.

Figure 8

Venn diagram detailing the number of proteins identified in eluates from peptide 6027 (white), peptide 21,358 (light grey) or mock (dark grey) columns loaded with cell-free extracts from T. gondii RH tachyzoites or mouse spleens. The complete datasets are given in Tables S1 and S2, respectively.

Figure 9

Protein–protein interaction network of proteins specifically binding to peptide 6027 in cell-free extracts of T. gondii RH tachyzoites based on the list of proteins given in Table S3. Two major clusters, namely ATP-synthase (blue ellipse) and the 26S-proteasome (red rectangle). are highlighted. The interaction network was created by the STRING knowledgebase and software tool from the Swiss Institute of Bioinformatics (www.expasy.org).

Figure 10

Protein–protein interaction network of proteins specifically binding to peptide 6027 in cell-free extracts of mouse spleens. Five clusters, namely cell division (blue), RNA processing (red), protein trafficking and antigen processing (yellow), erythrocyte and platelet surface (black), golgi apparatus (pink) and respiratory chain complex I (brown) are surrounded by ellipses. The interaction network was created by the STRING knowledge base and software tool from the Swiss Institute of Bioinformatics (www.expasy.org).