Activity of the Novel Peptide Arminin against Multiresistant Human Pathogens Shows the Considerable Potential of Phylogenetically Ancient Organisms as Drug Sources

Experiments were carried out with Hydra vulgaris strain AEP and Hydra magnipapillata strain 105. Transgenic animals were generated by using H. vulgaris strain AEP. The animals were cultured by standard procedures at 18°C.

The following microorganisms were used in this study: Bacillus megaterium ATCC 14581, Staphylococcus aureus ATCC 12600, MRSA ATCC 33393, MRSA ATCC 43300, MRSA 344, MRSA 355, MRSA 358, vancomycin-resistant strain Enterococcus faecalis ATCC 51299, vancomycin-resistant strain Enterococcus faecium 354, vancomycin-resistant strain Enterococcus faecium 356, vancomycin-resistant strain Enterococcus faecium G70, vancomycin-resistant strain Enterococcus faecium G71, ESBL-producing strain Klebsiella pneumoniae ATCC 700603, ESBL-producing strain Klebsiella pneumoniae CF1, ESBL-producing strain Klebsiella pneumoniae CF7, Escherichia coli Rosetta (Novagen), Escherichia coli DH5α, ESBL-producing strain Escherichia coli E4, and ESBL-producing strain Escherichia coli E9.

Nucleotide, protein, and translated BLAST engines at the NCBI server (1), Compagen (12), and http://hydrazome.metazome.net/cgi-bin/gbrowse/hydra/wereused for homology searches. The ClustalW program (6) was used for sequence alignments. The SignalP program was used to predict a possible signal peptide (4).

In situ hybridization was performed with H. magnipapillata strain 105, as described previously (2). The digoxigenin-labeled probe used covers the whole open reading frame of the gene for arminin 1a.

Recombinant c-arminin 1a was produced as a fusion protein and consisted of the SUMO protein (SMT3; Saccharomyces cerevisiae [21]) and the C-terminal part of arminin 1a starting with amino acid residue K58. The fusion construct was ligated into pPICHOLI (MoBiTec). The resulting overexpression construct contains a His tag at the N terminus. After the transformation of E. coli Rosetta (Novagen), the resulting clones were grown overnight on low-salt Luria-Bertani (LSLB; 2.5 g/liter NaCl) agar plates containing zeocin (25 μg/ml) and chloramphenicol (34 μg/ml). For each overexpression construct, a fresh clone was picked and grown in 5 ml LSLB medium containing zeocin (25 μg/ml) and chloramphenicol (34 μg/ml) overnight. The overnight culture was diluted 1:100 and was incubated at 37°C for 3 h until it reached an optical density at 600 nm (OD₆₀₀) of 0.6. Protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 2 mM. Three hours after induction, the bacteria were harvested by centrifugation at 8,000 × g for 10 min and were stored at −80°C. The bacterial pellet was resuspended in 50 mM sodium phosphate buffer, pH 8.0-300 mM NaCl. The suspension was sonicated on ice by using a burst regimen consisting of 10 15-s bursts, with a 15-s cooling period being used between each burst. After centrifugation at 15,000 × g for 30 min, the supernatants were applied to Ni-TED columns (Macherey & Nagel). The columns were washed, and the bound proteins were eluted with 250 mM levamisole in 50 mM sodium phosphate, pH 8.0-300 mM NaCl. The protein concentration was measured with a MicroBCA kit (Pierce). Subsequently, the fusion protein (SUMO-c-arminin 1a) was cleaved by using a SUMO protease kit (Invitrogen). One unit of SUMO protease per 20 μg of fusion protein was used in the digestion reaction, which was performed in 50 mM Tris-HCl, pH 8.0-150 mM NaCl-0.2% Igepal (Nonidet P-40)-1 mM dithiothreitol for 2 to 3 h at 30°C. The complete digestion mixture was subjected to reverse-phase high-pressure liquid chromatography (Äkta purifier; GE Healthcare) with a Vydac C₄ column (particle size, 5 μm; 250 mm by 3.2 mm; Grace) with gradient elution starting with 0.1% trifluoroacetic acid (TFA)-1.7% (vol/vol) acetonitrile to 0.1% TFA-59% (vol/vol) acetonitrile (slope, 1% [vol/vol] acetonitrile). Cleaved c-arminin 1a was eluted at 35.3% acetonitrile. These fractions were pooled, dried, and redissolved in 0.01% TFA and further analyzed.

A C-terminally amidated variant of c-arminin 1a (amino acid residues 58 to 88) was chemically synthesized as a trifluoroacetate salt at Caslo Laboratory ApS (Technical University of Denmark) and had a purity of 99.8%.

The antimicrobial activity of c-arminin 1a was evaluated as described previously (26). Briefly, test isolates were grown for 2 to 3 h in brain heart infusion broth at 36°C, washed three times in 10 mM sodium phosphate buffer (pH 7.4), supplemented with 1% tryptic soy broth, and adjusted to 10⁴ to 10⁵ bacteria/ml. A 100-μl volume of the bacterial suspension was mixed with 10 μl of c-arminin 1a solution (range of final concentrations tested, 0.0125 to 100 μg/ml) and incubated at 36°C. After 2 h, the numbers of CFU were determined. Bacterial suspensions supplemented with 10 μl of phosphate buffer-1% tryptic soy broth instead of c-arminin 1a served as negative controls. The results are given either as the 90% lethal doses or as the minimal bactericidal concentrations (MBCs; >99.9% killing).

The antimicrobial activity of c-arminin 1a against B. megaterium ATCC 14581, E. coli DH5α, and S. aureus ATCC 12600 in the presence of different salt concentrations was examined by the microdilution susceptibility assay described by Fedders and Leippe (8). The experiments were performed in 10 mM sodium phosphate buffer, pH 7.4, containing sodium chloride at a concentration of 0, 75, 150, or 200 mM. The activity in the presence of 0 mM sodium chloride was set equal to 1. The values were expressed as the mean values (including the standard deviations) of two independent experiments, each of which was performed in duplicate.

The hemolytic activities of c-arminin 1a and the cytotoxic peptide melittin (Sigma) were determined with human red blood cells, as described by Fedders and Leippe (8). In brief, peptides from stock solutions were serially diluted twofold in 50 mM sodium phosphate-150 mM NaCl, pH 7.4, starting from 250 μM for c-arminin 1a and from 20 μM for melittin. As controls, erythrocytes were incubated with distilled water (maximal lysis), and the buffer was supplemented with the corresponding volume of peptide solvent (spontaneous lysis). The absorbance of the diluted supernatants was measured at 405 nm. The values were expressed as the means of two independent experiments, each of which was performed in duplicate.

An overnight culture (12 to 16 h) of S. aureus ATCC 12600 was diluted 1:1,000 and was grown until an OD₆₀₀ of 0.1 was reached. The bacteria were washed twice with 10 mM sodium phosphate buffer, pH 7.4, supplemented with 1% tryptic soy broth and were concentrated to an OD₆₀₀ of 3.0 (∼10⁸ bacteria/ml). The bacterial suspension was incubated with c-arminin 1a at a final concentration of 41 or 410 μM peptide (100 and 1,000 times the MBC, respectively) in a final volume of 100 μl. The incubations were performed for 1.5 h at 37°C on a rocking platform. As a negative control, bacteria were incubated with solvent only. As a growth control, after the incubation, 50 μl of the bacterial suspension was plated on Luria-Bertani agar and the numbers of CFU per ml were determined. The other part of the treated bacterial suspension was fixed for 2 h at a ratio of 1:1 with 10% glutaraldehyde (Fluka) in 10 mM sodium phosphate buffer, pH 7.8. Subsequently, samples were treated and embedded as described previously (16).

To identify the genes involved in the innate immunity of Hydra magnipapillata, we previously produced cDNA libraries enriched for genes with functions in the immune response by using suppression subtraction hybridization (5). While 65% of the cDNA sequences obtained from this suppression subtraction hybridization showed matches to previously identified proteins by analysis with the BLAST program (5), the remaining 35% of the protein-encoding sequences showed no matches in any database and therefore might represent novel genes. To detect new AMPs within this fraction, we screened for short, secreted, and positively charged peptides. One of the genes which fulfilled these criteria had a strongly positively charged C-terminal region (pI > 12) and a predicted 18-amino-acid residue signal peptide sequence (Fig. 1a). The signal peptide and the cationic C-terminal region are separated by a highly negatively charged N-terminal region (pI < 4). Since detailed searches of other databases for conserved domains or orthologous sequences in genera other than Hydra failed to identify related genes, this sequence encodes a novel gene (Fig. 1a). We have termed this gene and peptide arminin 1a (Fig. 1). Analysis of the Hydra magnipapillata expressed sequence tags as well as the corresponding genome (http://hydrazome.metazome.net/cgi-bin/gbrowse/hydra) indicated that in addition to arminin 1a, there are several more related genes in Hydra magnipapillata (Fig. 2). According to their sequence homology, they can be grouped into three classes (Fig. 2). The two genes most closely related to arminin 1a, termed arminin 1b and arminin 1c, as well as arminin class 2 and class 3 genes, could represent isoforms which originated from recent gene duplication events. Interestingly, sequence alignment of all eight arminin-related peptides revealed that all peptides contain the positively charged C-terminal region. This region is rather nonconserved, whereas the negatively charged N-terminal part shows a high degree of conservation. It is known that a significant part of AMPs are made as precursors. Furthermore, there are examples, e.g., clavanins from Styela clava (29), ranalexin from Rana catesbeiana (7), and human defensin 5 (15), in which the active cationic part is released by cleavage from an anionic region. Unfortunately, there is no algorithm that can be used to predict the cleavages of antimicrobial propeptides. We therefore predicted that the active peptide in the C-terminal region was the longest possible fragment containing no negatively charged residues (c-arminin 1a). Starting with lysine (K58), c-arminin 1a has 31 amino acid residues and a high pI value (Fig. 1), and in addition, it consists of the region with the highest degree of variability among all Hydra arminins (Fig. 2, gray box). In contrast to the majority of AMPs isolated so far (18), c-arminin 1a and arminin 1a do not contain any cysteine residues.

Interestingly, in situ hybridization showed that arminin 1a is expressed in the entodermal epithelium along the body column (Fig. 1b). Since it was shown that the entoderm is the most vulnerable tissue in Hydra (5), this would be the most preferable site of expression of an AMP.

To test whether the predicted peptide from the C-terminal part of arminin 1a has antimicrobial activity, we produced recombinant c-arminin 1a in Escherichia coli. When c-arminin 1a was used in liquid growth inhibition assays, we observed a very strong bactericidal activity against Bacillus megaterium ATCC 14581, Escherichia coli DH5α, and Staphylococcus aureus ATCC 12600, indicating that this peptide is a potent component of the Hydra host defense. Uncovering a high degree of bactericidal activity against S. aureus led us to characterize the MBC against S. aureus in more detail. Additionally, it was interesting to see whether the already implied broad-spectrum efficacy against gram-negative and gram-positive bacteria holds true. For this larger screening, synthetically produced c-arminin 1a, which was amidated at the C terminus, was used. We confirmed (Table 1) that c-arminin 1a is highly active against B. megaterium ATCC 14581, E. coli DH5α, and S. aureus ATCC 12600. In addition, c-arminin 1a was found to be capable of killing a number of MRSA strains at very low concentrations of 0.4 to 0.8 μM (Table 1). Moreover, c-arminin 1a was also active against gram-positive vancomycin-resistant Enterococcus faecalis and Enterococcus faecium strains. Interestingly, c-arminin 1a was able to kill gram-negative ESBL-producing members of the Enterobacteriaceae family, such as K. pneumoniae strains and E. coli strains E4 and E9 (Table 1). These observations make c-arminin 1a a potent and broad-spectrum AMP and therefore an interesting lead structure for the design of a novel group of antibiotics.

To assess the activity of c-arminin 1a in the presence of different salt concentrations, we used a microdilution susceptibility assay and NaCl concentrations ranging from 0 to 200 mM. As shown in Fig. 3a, the activity of c-arminin 1a against E. coli DH5α and S. aureus ATCC 12600 in the presence of a salt concentration that mimics the physiology of human blood (150 mM) decreased by only a factor of 2, while the activity against B. megaterium ATCC 14581 was more than four times higher than that under low-salt conditions. A further increase in the salt concentration did not significantly affect the activity of c-arminin 1a against E. coli and B. megaterium, while the activity against S. aureus continued to decrease slightly. These data show that c-arminin 1a is still active even under the physiological conditions of human blood. That finding was unexpected, since Hydra is an obligate freshwater animal with nearly no salt tolerance.

Selectivity, or the killing of bacteria at concentrations not harmful to normal eukaryotic cells, is a highly desirable characteristic of an antimicrobial agent. To determine selectivity, c-arminin 1a was evaluated for its toxicity to human red blood cells by using an erythrocyte lysis assay under the physiological conditions of human blood. Red blood cells were incubated with various concentrations of c-arminin 1a in 10 mM phosphate buffer for 1 h. As shown in Fig. 3b, even at concentrations 50 times higher then the MIC, no harmful effects of c-arminin 1a on red blood cells could be detected. As a control, the exposure of red blood cells to the cytotoxic peptide melittin resulted in dose-dependent hemolytic activity.

In general, AMPs have different ways of interfering with the mechanisms essential for bacterial survival. The most common and best-studied mode is the integration of AMPs into the bacterial membrane, which leads to membrane destabilization, energy leakage, and finally, the death of the bacterial cell. To understand how exposure to c-arminin 1a leads to the death of the bacterial cell, we examined the morphological changes to S. aureus ATCC 12600 (10⁸ CFU/ml) after treatment with 410 μM of c-arminin 1a for 1.5 h by transmission electron microscopy. As shown in Fig. 4, short-term exposure to arminin 1a leads to the destruction and detachment of the peripheral cell wall of S. aureus ATCC 12600. S. aureus cells exposed to the AMP human beta defensin 3 (HBD3) showed similar cell wall defects (11). Interestingly, HBD3 and c-arminin 1a also share other functional characteristics, such as salt insensitivity and broad-spectrum antimicrobial activity, especially against MRSA and VRE strains (11). Since it is known that HBD3 interferes with the membrane-bound enzyme machinery for cell wall biosynthesis (27), it will be interesting to find out whether c-arminin 1a functions in a similar manner.

In the last 2 years, evidence has accumulated that the innate defense systems of ancient organisms employ both evolutionarily conserved signaling pathways and novel taxon-specific host defense-associated molecules (3, 5, 13, 16, 23). Thus, despite the conservation of the pathogen recognition system and the signaling pathways (5, 25), effector molecules have been modified early during animal evolution and branched off into a variety of unique components in present-day organisms. We have proposed elsewhere (17) that such effector molecules without homologues, classified as orphan or taxonomically restricted genes, in combination with the rewiring of the conserved regulatory networks, allow organisms to make fine adaptations to constantly changing ecological conditions. Why do novel AMPs isolated from organisms only very distantly related to humans and living in completely different environments kill bacteria pathogenic for humans with such a high degree of efficacy? Colonizing microbiota are known to adapt quickly to their hosts (9, 10). Therefore, bacteria closely associated with a particular species may be much less sensitive to the antimicrobial molecules produced by their hosts than to the antimicrobial molecules derived from unrelated organisms.

Taken together, ancient animals such as Hydra and their plethora of highly active novel AMPs appear to be sources of antimicrobial drugs that are effective even against multiresistant bacteria.