Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

In this study, we investigated the antibacterial activities of DIF derivatives in vitro and show that several of them exert strong antibacterial effects against Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and, Enterococcus faecium. In addition, several of the DIF derivatives evaluated strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant enterococci (i.e., strains of E. faecalis and E. faecium). Our results support the investigation of such DIF derivatives as candidate lead compounds for developing novel antimicrobials.
In this study, we investigated the antibacterial activities of DIF derivatives in vitro and show that several of them exert strong antibacterial effects against Gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and, Enterococcus faecium. In addition, several of the DIF derivatives evaluated strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant enterococci (i.e., strains of E. faecalis and E. faecium). Our results support the investigation of such DIF derivatives as candidate lead compounds for developing novel antimicrobials.
In addition, a diffusion assay without paper discs was performed for comparison. Briefly, 10 mL of S. aureus in MHB (10 7 CFU/mL) was spread on an MHB agar plate (diameter, 100 mm), after which 2 µL of each 10 mM DIF solution was dropped directly on the agar surface. The plates were incubated for 20 h at 37 • C, after which the bacterial growth-inhibition zones around the discs were noted.

Measurement of Minimum Inhibitory Concentration
Gram-positive and Gram-negative bacteria in MHB (5 × 10 5 CFU/mL; 0.1 mL/well) were incubated for 20 to 24 h at 37 • C in 96-well plates (Corning, NY, USA) in the presence of vehicle, various concentrations of serially diluted DIF derivatives, or known antibiotics; MIC was defined as the lowest concentration of the additives that inhibited visible bacterial growth.

Transmission Electron Microscopy
Specimens for transmission electron microscopy were prepared according to standard procedures [32]. Briefly, S. aureus (strain ATCC29213) in MHB (1-5 × 10 8 CFU/mL; 2 mL per well) was incubated for 1.5 h at 37 • C in 6-well plates (Corning) in the presence of 0.2% DMSO (vehicle) or 4 µM DIF derivative. After incubation, the bacteria were transferred to centrifuge tubes, collected by centrifugation (4000 × g, 1 min), fixed overnight at 4 • C in 0.5 mL of 2.5% (v/v) glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (LSI Medience Corporation, Tokyo, Japan), and postfixed in 1% (w/v) OsO 4 in 100 mM phosphate buffer. Fixed specimens were dehydrated through a graded series of ethanol and embedded in Epon812 (Oken-Shoji, Tokyo, Japan). Ultrathin sections were cut by using an ultramicrotome (model UC6; Leica, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. These sections were examined under a transmission electron microscope (model HT7700; Hitachi High-Tech, Tokyo, Japan).

Antimicrobial Effects of DIFs and Their Derivatives on S. aureus in Agar Plates
We first examined the effects of DIF-1, DIF-3 ( Figure 1A), and their derivatives ( Figure 1B,C) on the growth of methicillin-susceptible S. aureus (MSSA: strain 209P) ( Figure 2A) and methicillin-resistant S. aureus (MRSA: strain MS29202) ( Figure 2B) by using a paper-disc diffusion assay. In MSSA (Figure 2A), a large ring free of bacterial growth formed around the disc containing penicillin and streptomycin, as expected. Similar growth-free rings developed around the discs containing DIF-1 and several DIF-1 derivatives but not DIF-3. In MRSA-containing plates ( Figure 2B), penicillin and streptomycin failed to inhibit bacterial growth. However, DIF-1 and several DIF-1 derivatives, but not DIF-3, again produced zones free of bacterial growth. Although the MRSA strain we used was susceptible to minomycin, it was resistant to imipenem, levofloxacin, and clindamycin and was marginally resistant to gentamicin ( Figure 2C). These results indicate that various DIFs and their derivatives possess antimicrobial activity against S. aureus. In addition, the mechanism underlying the antimicrobial action of DIFs likely differs (at least in part) from those of the known antibiotics that we used.

Antimicrobial Effects of DIFs and Their Derivatives on S. aureus in Agar Plates
We first examined the effects of DIF-1, DIF-3 ( Figure 1A), and their derivatives ( Figure 1B, 1C) on the growth of methicillin-susceptible S. aureus (MSSA: strain 209P) ( Figure 2A) and methicillin-resistant S. aureus (MRSA: strain MS29202) ( Figure 2B) by using a paper-disc diffusion assay. In MSSA (Figure 2A), a large ring free of bacterial growth formed around the disc containing penicillin and streptomycin, as expected. Similar growth-free rings developed around the discs containing DIF-1 and several DIF-1 derivatives but not DIF-3. In MRSA-containing plates ( Figure  2B), penicillin and streptomycin failed to inhibit bacterial growth. However, DIF-1 and several DIF-1 derivatives, but not DIF-3, again produced zones free of bacterial growth. Although the MRSA strain we used was susceptible to minomycin, it was resistant to imipenem, levofloxacin, and clindamycin and was marginally resistant to gentamicin ( Figure 2C). These results indicate that various DIFs and their derivatives possess antimicrobial activity against S. aureus. In addition, the mechanism underlying the antimicrobial action of DIFs likely differs (at least in part) from those of the known antibiotics that we used. Paper discs were placed on Mueller-Hinton broth (MHB) agar plates that contained MSSA (209P); paper discs were impregnated with 2 µL of either ethanol (EtOH; vehicle), DMSO (vehicle), a 10 mM solution of the indicated DIF or DIF derivative, or a mixture of penicillin (5000 units/mL) and streptomycin (5 mg/mL). After incubation for 20 h at 37 °C, bacterial growth-inhibition zones around the discs were noted. (B, C) Effects of DIF derivatives on the growth of methicillin-resistant S. aureus (MRSA) on agar. Paper discs were placed on MHB agar plates that contained MRSA (MS29202); these discs were impregnated with 2 µL of either EtOH (vehicle), DMSO (vehicle), a 10 mM solution of the indicated DIF or DIF derivative, or a mixture of penicillin and streptomycin (B). In addition, paper discs containing the indicated antibiotics were placed on Paper discs were placed on Mueller-Hinton broth (MHB) agar plates that contained MSSA (209P); paper discs were impregnated with 2 µL of either ethanol (EtOH; vehicle), DMSO (vehicle), a 10 mM solution of the indicated DIF or DIF derivative, or a mixture of penicillin (5000 units/mL) and streptomycin (5 mg/mL). After incubation for 20 h at 37 • C, bacterial growth-inhibition zones around the discs were noted. (B, C) Effects of DIF derivatives on the growth of methicillin-resistant S. aureus (MRSA) on agar. Paper discs were placed on MHB agar plates that contained MRSA (MS29202); these discs were impregnated with 2 µL of either EtOH (vehicle), DMSO (vehicle), a 10 mM solution of the indicated DIF or DIF derivative, or a mixture of penicillin and streptomycin (B). In addition, paper discs containing the indicated antibiotics were placed on MRSA agar plates (C). After incubation for 20 h at 37 • C, bacterial growth-inhibition zones around the discs were noted.

MIC Values of DIF Derivatives in S. aureus, B. subtilis, E. coli, and M. bovis
We next examined the effects of DIF derivatives on the growth of the Gram-positive bacteria MSSA (strain 209P), MRSA (MS29202), and B. subtilis (ATCC6633) and on the Gram-negative bacterium E. coli (NIHJ), and MIC values of DIF derivatives were determined ( Table 1). Several of the DIF derivatives showed strong antibacterial activity against the Gram-positive bacteria, yielding MIC values of less than 1 µM. However, none of the DIF derivatives that we tested inhibited the growth of E. coli and M. bovis (MIC, >100 µM) ( Table 1); M. bovis was sensitive to the antibiotics tiamulin (MIC, 0.5 µM) and enrofloxacin (0.7 µM). Note that the MIC values of DIF-3 against MSSA and MRSA were comparable to those of DIF-1 (Table 1), even though DIF-3 lacked antibacterial activity against these strains in the paper-disc diffusion assay ( Figure 2). These results suggest that DIF-3 and its derivatives exert antibacterial activity in solution but become ineffective on solid media (e.g., paper disc, agar). To examine this hypothesis, we compared the effects of DIF-1, DIF-3, and three DIF-3 derivatives on the growth of MSSA and MRSA by using DIF-impregnated paper discs or by spotting the DIF solutions directly on the surface of the bacteria-containing agar (Figure 3). Again, DIF-1-but not DIF-3 or its derivatives-showed a strong antibacterial activity against MSSA and MRSA in the paper-disc diffusion assay ( Figure 3A,C).
However, when the solutions were dropped directly on the agar, all of the DIF derivatives we tested showed antibacterial activity against the bacteria ( Figure 3B,C). These results suggest that DIF-3 and its derivatives are likely to diffuse poorly through some matrixes, including agar and paper.
Biomolecules 2019, 9, x 6 of 12 MSSA and MRSA by using DIF-impregnated paper discs or by spotting the DIF solutions directly on the surface of the bacteria-containing agar ( Figure 3). Again, DIF-1-but not DIF-3 or its derivatives-showed a strong antibacterial activity against MSSA and MRSA in the paper-disc diffusion assay ( Figure 3A,C). However, when the solutions were dropped directly on the agar, all of the DIF derivatives we tested showed antibacterial activity against the bacteria ( Figure 3B,C). These results suggest that DIF-3 and its derivatives are likely to diffuse poorly through some matrixes, including agar and paper.

MIC Values of DIF Derivatives in S. aureus, E. faecalis, and E. faecium
We then further assessed the potential antibacterial activities of representative DIF derivatives (Figure 1) on the growth of MSSA (strain ATCC29213), MRSA (ATCC43300), VSE (ATCC29212), and VREs (ATCC51299 and ATCC700221); the latter two strains carry the vancomycin-resistant genes vanB and vanA, respectively (Table 2). Again, several DIF derivatives showed strong antibacterial activity against MSSA and MRSA (S. aureus strains different from those in Table 1), with MIC values of less than 2 µM (Table 2). In addition, various DIF derivatives demonstrated strong antibacterial effects against VSE and two VRE strains, with associated MIC values of less than 2 µM ( Table 2); note that the MRSA strain was highly resistant to cefoxitin and oxacillin, and the two VREs were highly or intermediately resistant to vancomycin and ampicillin ( Table 2).

MIC Values of DIF Derivatives in S. aureus, E. faecalis, and E. faecium
We then further assessed the potential antibacterial activities of representative DIF derivatives (Figure 1) on the growth of MSSA (strain ATCC29213), MRSA (ATCC43300), VSE (ATCC29212), and VREs (ATCC51299 and ATCC700221); the latter two strains carry the vancomycin-resistant genes vanB and vanA, respectively (Table 2). Again, several DIF derivatives showed strong antibacterial activity against MSSA and MRSA (S. aureus strains different from those in Table 1), with MIC values of less than 2 µM (Table 2). In addition, various DIF derivatives demonstrated strong antibacterial effects against VSE and two VRE strains, with associated MIC values of less than 2 µM ( Table 2); note that the MRSA strain was highly resistant to cefoxitin and oxacillin, and the two VREs were highly or intermediately resistant to vancomycin and ampicillin ( Table 2). These results indicate that DIF derivatives may possess strong antibacterial activities against a broad range of Gram-positive bacteria, including known drug-resistant bacteria such as MRSAs and VREs.

Effects of DIF Derivatives on the Ultracellular Structure of S. aureus
To help elucidate the mechanism underlying the antibacterial activity of DIF derivatives, we examined the effects of Ph-DIF-1, Ph-DIF-3, and Bu-DIF-3 on the ultracellular structure of MSSA (strain ATCC29213). Transmission electron microscopy revealed distinct multilayered structures consisting of cell wall and/or plasma membrane in the DIF-treated MSSA cells (Figure 4). This finding suggests that the tested DIF derivatives suppress the growth of S. aureus by hindering cell wall formation.
When we assessed the MIC values of these dichlorinated dibenzofurans in MRSA (strain MS29202), neither AB0022A nor Pf-2 had any noteworthy inhibitory effect (MIC, >100 µM) on the growth of this strain ( Figure 5B). In contrast, Pf-1 suppressed the growth of this MRSA at a MIC value of 12.5 µM, which was comparable to those of DIF-1 and DIF-3 (25 and 12.5 µM, respectively). The results suggest that DIF derivatives such as CP-DIF-3 and Ph-DIF-3 (Tables 1 and 2) are likely better lead antibacterial drugs than the chlorinated dibenzofurans we evaluated.

Discussion
Most of the many antibiotics (antimicrobials) identified since the discovery of penicillin in the fungus Penicillium notatum have been isolated from fungi and Actinobacteria [33][34][35][36]. However, due to intensive use, overuse, or the use of subtherapeutic concentrations of antibiotics, the number of drug-resistant bacteria, such as MRSAs and VREs, is growing. A search for new antibiotic molecules and bio-resources that produce novel antimicrobials is required [9,[35][36][37]. which was comparable to those of DIF-1 and DIF-3 (25 and 12.5 µM, respectively). The results suggest that DIF derivatives such as CP-DIF-3 and Ph-DIF-3 (Tables 1 and 2) are likely better lead antibacterial drugs than the chlorinated dibenzofurans we evaluated.

Discussion
Most of the many antibiotics (antimicrobials) identified since the discovery of penicillin in the fungus Penicillium notatum have been isolated from fungi and Actinobacteria [33][34][35][36]. However, due to intensive use, overuse, or the use of subtherapeutic concentrations of antibiotics, the number of drug-resistant bacteria, such as MRSAs and VREs, is growing. A search for new antibiotic molecules and bio-resources that produce novel antimicrobials is required [9,[35][36][37].
The cellular slime mold D. discoideum has long been used as a model organism in the study of cell and developmental biology. However, slime molds have recently been identified as excellent sources of potential lead compounds for drug discovery and the development of novel medicines [9,38]. In this regard, we have focused on elucidating the biologic and pharmacologic activities (such as antitumor and antiparasitic effects) of the D. discoideum-derived DIFs and their derivative compounds in various types of eukaryotic cells [9,16,29].
In the present study, we showed that several D. discoideum DIF derivatives possess antibacterial activity against Gram-positive bacteria, including S. aureus, E. faecalis, E. faecium, and B. subtilis. In contrast, these DIF derivatives did not inhibit the growth of Mycoplasma or the Gram-negative bacterium E. coli (Tables 1 and 2). Most importantly, several of the DIF derivatives we evaluated strongly suppressed the growth of MRSAs and VREs (Tables 1 and 2). Note that the MRSA strain MS29202 was resistant not only to penicillin and streptomycin ( Figure 2B) but also to clindamycin, levofloxacin, and imipenem ( Figure 2C), whereas the MRSA strain (ATCC43300) was resistant to both cefoxitin and oxacillin (Table 2). In addition, the VRE isolates E. faecalis (which harbors the vancomycin-resistance gene vanB) and E. faecium (containing vanA) were resistant to both vancomycin and ampicillin (Table 2). Although they are primarily opportunistic pathogens, E. faecalis and E. faecium cause the great majority of enterococcal infections, and isolates that carry drug-resistance genes such as vanA or vanB can cause serious infections [39,40]. Overall, our results suggest that the mechanism underlying the antibacterial actions of DIF derivatives likely differ (at least in part) from those of the known antibiotics we assessed. Consequently, DIF derivatives are promising lead compounds for novel antimicrobials against a broad range of Gram-positive bacteria, including known drug-resistant strains.
Although the antibacterial activities of the DIF derivatives varied among the Gram-positive strains of bacteria examined in this study (Tables 1 and 2), the structure-activity relationship analysis revealed that compounds with longer alkyl chains at the acyl group (e.g., DIF-3(+2) and DIF-3(+3)) and compounds with larger alkyl groups in place of the methyl group (e.g., Bu-DIF-3, Hex-DIF-3, and Ph-DIF-3) had greater antibacterial activities against Gram-positive strains than did those with shorter alkyl chains or smaller alkyl groups, and that compounds with one chlorine substituted in the benzene ring had greater antibacterial activities against Gram-positive strains than did those with two chlorines substituted in the benzene ring. In contrast, there was no clear overall relationship between hydrophobicity (assessed as ClogP value) and antibacterial activity (Table 1).
Regarding the mechanism through which DIF derivatives exert their antibacterial effects, DIF derivatives have mitochondrial uncoupling activity in mammalian cells [17,18]. Given that mitochondria and bacteria are similar in many aspects, these previous findings suggest that DIF derivatives may affect bacterial proton transport (or intracellular pH) and thereby suppress bacterial growth. However, the strength of the mitochondrial uncoupling properties of DIF derivatives [17,18] does not necessarily correlate with the strength of their antibacterial activities (Tables 1 and 2). Furthermore, in the present study, we showed that in S. aureus, Ph-DIF-1, Ph-DIF-3, and Bu-DIF-3 induce the formation of distinct multilayered structures composed of cell wall or membrane (Figure 4). Although the relationship between the antibacterial effects of DIFs and the DIF-induced formation of these structures is unclear currently, this observation may be a clue for elucidating the mechanism underlying the antibacterial activity of DIF derivatives.

Conclusions
In this study, we showed that several derivatives of DIF-1 and DIF-3, chlorinated polyketides found in D. discoideum possess strong antimicrobial activities against Gram-positive bacteria including MRSAs and VREs. Our results suggest that the DIF derivatives are good lead compounds for developing novel antimicrobials.

Patents
The following authors hold a patent related to this article: Kubohara