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Figure legends

550

Fig. 1. Phylogenetic tree for the N25, N6, N12, N18 isolates based on 16S rRNA gene

551

sequences. The maximum-likelihood method based on the Tamura-Nei model was used.

552

Bootstrap values are based on 1,000 replicates. The 16S rRNA gene sequence from

553

Actinomadura hibisca (NR_042031) was used as the outgroup.

554

555

Fig. 2. (A) Structure of 1-phenazinecarboxylic acid. (B) LC-HRMS analysis of the active

556

compound in F8. The upper plot shows the total ion chromatogram (TIC), total scan

557

photodiode array chromatogram and extracted ion chromatogram from m/z 225.0661±5

558

ppm (m/z 225.0650-225.0672) (shown in order from the top). The middle and lower plots

559

represent the ultraviolet (UV) spectrum and mass spectrum, respectively, each with a

560

retention time (Rt) of 2.95 min.

561

562

Fig. 3. (A) Structure of 1-phenazinecarboxylic acid-methyl ester. (B) LC-HRMS analysis

563

of the active compound in F9. The upper plot shows the total ion chromatogram (TIC),

564

total scan photodiode array chromatogram and extracted ion chromatogram from m/z

565

239.0819±5 ppm (m/z 239.0807-239.0831) (shown in order from the top). The middle and

566

lower plots represent the ultraviolet (UV) spectrum and mass spectrum, respectively, each

567

with a retention time (Rt) of 3.09 min.

568

569

Fig. 4. (A) Anti-Toxoplasma activity of phenazine-1carboxylic acid (PCA) based on the T.

570

gondii RH-GFP strain. The half maximal inhibition concentration (IC50) was 55.5 µg/ml.

26

571

(B) Antimalarial activity of PCA based on the P. falciparum 3D7 strain. The IC50 value

572

was 6.4 µg/ml. Data represent the mean values ± SD for three independent experiments.

573

27

Table 1.

Anti-Toxoplasma and antimalarial activities of antibiotics derived from Streptomyces species.

Antibiotics

Actinomycetes

IC50 for T.

IC50 for P.

falciparum

gondii

IC50 for HFF

cells

(Selectivity index)

Streptomyces

Amphomycin [7]

>100 µM

9.3 µM

>100 µM

>100 µM

>100 µM

11.5 µM

>100 µM

>100 µM

1.2 µM

>100 µM

182.7 µM

>100 µM

>100 µM

34.1 nM (1.0)

33.1 nM

canus

Streptomyces

Kanamycin [35]

canus

Resistomycin

Streptomyces

[36]

canus

Tylosine

Streptomyces

phosphate [14]

cirratus

Bafilomycin A1

Streptomyces

[39]

bacillaris

All IC50 values were calculated based on three independent experiments.

Table 2

Anti-Toxoplasma and antimalarial activities of crude extracts from four actinomycetes (N6,

N12, N18, N25) isolates.

IC50 for T. gondii

IC50 for P. falciparum

(Selectivity index)

(Selectivity index)

N6

>100 µg/ml

>100 µg/ml

>100 µg/ml

N12

>100 µg/ml

>100 µg/ml

>100 µg/ml

N18

91.8 ng/ml (3.4)

156.9 ng/ml (2.0)

316 ng/ml

N25

19.2 µg/ml

4.9 µg/ml

>100 µg/ml

Actinomycetes

All IC50 values were calculated based on three independent experiments.

IC50 for HFF cells

Table 3

Anti-Toxoplasma and antimalarial activities of the TLC fractions from Streptomyces canus

(N25).

IC50 for T. gondii

IC50 for P. falciparum

(Selectivity index)

(Selectivity index)

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

>50 µg/ml

>50 µg/ml

>100 µg/ml

2.6 µg/ml (53.4)

4.8 µg/ml (28.9)

138.9 µg/ml

122.2 ng/ml (239.7)

163.8 ng/ml (178.8)

29.3 µg/ml

10

>50 µg/ml

18.4 µg/ml

>100 µg/ml

Fractions

IC50 for HFF cells

>50 µg/ml: No activity at 50 µg/ml; the highest dose tested.

The IC50 values of fractions 8, 9, and 10 were calculated based on three independent

experiments.

Table S1

Hemolysis rate (%)

Drugs

Concentrations Hemolysis rate (%)

Amphomycin

100 µM

0.41

Bafilomycin A1

100 µM

7.78

Tylosine phosphate

100 µM

N.D.

Crude extract N18

100 µg/ml

3.01

Crude extract N25

100 µg/ml

0.41

Fraction 8

100 µg/ml

N.D.

Fraction 9

100 µg/ml

0.1

Fraction 10

100 µg/ml

6.02

PCA

100 µg/ml

N.D.

N.D.: Below detection limit

PBS and RBC lysis buffer (0.83% NH4Cl; 0.01 M Tris-HCl, pH 7.2) are used for negative and

positive controls, respectively.

Procedure: Hemolysis assay was performed as reported previously with minor modification

(Evans et al., 2013). Each drug, extract and fraction was prepared in PBS as designed

concentration and then 3% of erythrocyte suspension in PBS was added. The sample were

incubated at 37ºC for 3 h and then the mixtures were centrifuged at 1,300 × g for 5 min. A 100

µl of supernatant was transferred into 96-well plate. The absorbance values of supernatants

were determined with microplate reader at 540 nm. The hemolysis rate of erythrocytes was

calculated by using the following formula.

Hemolysis rate = (A sample  A negative control)/(B positive control  B negative control) 100%

Reference: B.C. Evans, C.E. Nelson, S. S. Yu, K.R. Beavers, A.J. Kim, H.LI, H.M. Nelson,

T.D. Giorgio, C.L. Duvall, Ex vivo red blood cell hemolysis assay for the evaluation of pHresponsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs, J Vis Exp.

73 (2013), e50166, doi: 10.3791/50166.

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