Human natriuretic peptides exhibit antimicrobial activity

Human natriuretic peptides exhibit antimicrobial activity

Alexander Krause, Cornelia Liepke, Markus Meyer, Knut Adermann, Wolf-Georg Forssmann and Erik Maronde

IPF PharmaCeuticals GmbH, Feodor-Lynen-Strasse-31, 30625 Hannover, Germany

Abstract Here we describe a novel function for members of the well-characterized human natriuretic peptide family. Human „brain-type natriuretic peptide“ (hBNP-32) as well as other members of this peptide class are antimicrobially active against Gram-positive and Gramnegative bacteria and yeast in a dose-dependent manner. This activity of natriuretic peptides is comparable to that of known antimicrobial peptides such as casocidins or magainins.

1. Introduction In vertebrates natriuretic peptides are ligands for the so-called particulate guanylate cyclase receptors which mediate a wide range of well-defined physiological effects [1,2]. These actions are mediated by an increase in the intracellular cGMP concentration [2]. Effects of natriuretic peptides mediated by the smaller clearance receptor, which is lacking an intracellular guanylate cyclase domain, involve a G-protein coupled signal transduction pathway [2,3]. To our knowledge nothing is known about an antimicrobial activity of natriuretic peptides. To date, many peptides from various origin like the defensins [4,5,6] or the milkderived casocidins [7, 8] are known to be antimicrobially active against a variety of pathogenic germs [4]. Moreover, only one vertebrate peptide hormone, somatostatin, has been demonstrated to inhibit bacterial growth in Helicobacter pylori so far [9]. We show here that members of the human natriuretic peptide family, with hBNP-32 as the most active compound, are antimicrobially active against both Gram-positive and Gramnegative bacteria and yeast. A putative mechanism of this effect of hBNP-32 could have been the presence of a bacterial homolog of a particulate guanylate cyclase which has not been described yet. If such a receptor existed, the hBNP-32 effect should have been also inducible by application of a cGMP-analog like 8-Br-cGMP. However, the effect of hBNP-32 was not mimicked by cGMP and may thus be mechanistically different to the previously described antimicrobial effect of somatostatin on Helicobacter pylori [9].

2. Experimental 2.1 Material Natriuretic peptides were both purchased from Sigma (Deisenhofen, Germany) and synthesized at IPF PharmaCeuticals according to established procedures [10]. Peptides from both sources gave similar results (see Fig.4).

2.2 Microorganisms Escherichia coli BL21 was obtained from J. Alves, Hannover Medical School. Staphylococcus carnosus was kindly provided by F. Goetz, University of Munich. Bacillus subtilis ATCC6051, Pseudomonas aeruginosa PAO1, Neisseria cinerea ATCC14685 and Saccharomyces cerevisiae ATCC9763 were purchased from the „Deutsche Sammlung für Zellkulturen und Mikroorganismen“ (DSMZ, Braunschweig, Germany).

2.3 Antimicrobial assays Antimicrobial activity was assessed essentially as described earlier [11,12]. The agarose layer consisted of 0.03% (w/v) Tryptic Soy Broth (TSB, Sigma, Deisenhofen, Germany) in 10 mM sodium phosphate buffer (pH 7.2) with 0.02% Tween 20 (v/v; Sigma) and 0.8% GTG agarose (FMC BioProducts, Rockland, USA). The plates were incubated at 37oC for 16 hours before the size of an inhibiton zone was recorded. Antimicrobial activity is expressed in inhibition units according to [11], with one unit corresponding to 0.1 mm diameter of growth inhibitory area.. A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of the peptides. Two-fold serial dilutions of peptides were prepared in test medium consisting 3 g/l TSB in 10 mM sodium phosphate buffer (pH 7.2). A standard inoculum of 50 µl of microorganisms in test medium containing 2 - 5 x 105 CFU/ml was

added to 50 µl of peptide solution. Following an incubation period of 16 to 20 hours at 37oC, bacterial growth was determined by visual analysis and absorbance measurement at 600 nm using a Dynatech ELISA reader. The MIC was taken as the lowest peptide concentration at which bacterial growth was inhibited after 18 + 2 hours. Subsequently, minimum bactericidal concentrations (MBC) were determined by transferring 99.9 µl from each clear well (

MIC)

to an individual well of a 24-well microtiter plate containing 1 ml of LB medium. After incubation for 20 h at 37 °C, the minimum bactericidal concentration was identified as the lowest peptide concentration that did not permit any visible bacterial growth.

2.4 Proteolytic cleavage Proteolytic cleavage was performed with subtilisin in 25 mM Tris-HCl buffer pH 7.0 at a protease to peptide ratio of 1:100 (w/w). The reactions were stopped by heating to 60°C for 5 min. Samples without enzyme were used as control [7,8].

2.5 Statistics Shown are the means + standard deviation calculated from the raw data using GraphPad Prism 3.0 software (GraphPad, San Diego, USA).

3. Results and Discussion In a screening for antimicrobially active peptides using a sensitive radial diffusion assay [11] we identified hBNP-32 (Fig.1,2 and 4) and other members of the natriuretic peptide family (Fig. 2 and 3) as active compounds against Gram-positive and –negative bacteria and yeast, whereas both parathyroid hormone 1-37 (PTH) and cortitropin releasing hormone (CRH) were inactive. The activity of hBNP-32 was dose-dependent and comparable to that of the known antimicrobial peptide casocidin-I, but weaker than the non-peptide antibiotic ampicillin (Fig.1). Casocidin-I is about as active as Magainin, a peptide antibiotic, whose analogs entered clinical phase III. The minimum inhibitory concentrations (MIC) of hBNP-32 were 19 µg/ml (=5.5 µM) for Staphylococcus carnosus, 19 µg/ml for Bacillus subtilis and 100 µg/ml for Escherichia coli BL21. The minimum bactericidal concentrations were 37.5 µg/ml (=10.8 µM) for Staphylococcus carnosus, 75 µg/ml for Bacillus subtilis and 200 µg/ml for Escherichia coli BL21, indicating a bactericidal rather than a bacteriostatic activity of hBNP-32. Moreover, hBNP-32 exhibited antimicrobial acitivity against the pathologically relevant bacterium Pseudomonas aeruginosa (MIC: 125 µg/ml). Such MIC and MBC values are well within the range for peptide antibiotics [12,13]. The antimicrobial activity of hBNP-32 from different sources was equivalent (Fig.4). The activity was destroyed after hydrolysis with the protease subtilisin indicating that the whole molecule or large parts of it rather than small fragments were responsible for this action (data not shown). The antimicrobial effect was not mimicked by application of the second messenger analogs 8-Br-cGMP or 8-Br-cAMP (Fig.1). This is indicative of a mechanism different from the growth inhibitory action of somatostatin on Helicobacter pylori which was reportedly also induced by 8-Br-cGMP application [9].

Many of the known antimicrobially active peptides show an excess of positively charged amino acids and are therefore cationic at physiological pH due to an abundance of basic arginine- and lysine residues, which is reflected in a high isoelectric point (pI). With respect to the pI or net positive charge, natriuretic peptides behave as described for other antimicrobial peptides, namely hBNP-32 (calculated pI 10.95), urodilatin (calculated pI 11.83) [14] and human atrial natriuretic peptide (hANP; calculated pI 11.83; Fig. 2,3). The least effective of the tested natriuretic peptides was human C-type natriuretic peptide (hCNP;calculated pI 8.9; Fig. 2). Thus, the more basic of the natriuretic peptides exhibited antimicrobial activity, whereas those comprising more acidic pIs (hCNP; also guanylin and uroguanylin, data not shown) were less active. Interestingly, rat BNP-32 (calculated pI 9.5), which is 50% identical to hBNP-32, had no detectable antimicrobial activity, whereas the long form of rat BNP, rBNP-45 (pI 10.07), was weakly active against B. subtilis (data not shown). In S. cerevisiae hBNP-32 was also the most active peptide, no matter if hBNP-32 was purchased (Fig.4a) or synthesized inhouse (Fig.4b). Another suspected property of antimicrobial peptides like defensins is their ability to form pores in bacterial membranes [4], a property that has interestingly been demonstrated for synthetic hCNP, the least efficient of the natriuretic peptides in respect to antimicrobiosis in the present study [15,16]. Most of the antimicrobial peptides have so far been discovered in non-human species [4,13], but recently there have been more hints that the human body produces a broad spectrum of substances involved in host defense, such as defensins [4], and the bulk of which is, together, responsible for the antimicrobial homeostasis. Our data show that the earlier characterized hBNP-32 exerts an antimicrobial function against a broad spectrum of germs. Beside its high expression in the heart the hBNP gene is expressed at lower levels (10 to 100fold lower) in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small

intestine, ovary, uterus, and striated muscle [17]. Expression in the lung and the small intestine is typical for other antimicrobial peptides as well [4]. Although overall expression may seem rather low in these tissues specialized cells inside such an organ can produce very high levels like the Paneth cells in the gut [4,13]. Furthermore, as hBNP-32 is strongly upregulated in different cardiac diseases [18], the relation to infectious elements in the course of these syndromes in the heart has to be considered.

References [1] Samson, W.K. and Levin, E.R. (eds.) (1997) Natriuretic peptides in health and disease p. 1-337. [2] Potter, L.R. and Hunter, T. (2001) J. Biol. Chem. 2001 276, 6057-6060. [3] Murthy, K.S., Teng, B.Q., Zhou, H., Jin, J.G., Grider, J.R. and Makhlouf, G.M. (2000). Am. J. Physiol. 278, G974-G980. [4] Schröder, J.M. (1998) Biochem. Pharm. 57, 121-134. [5] Bensch, K.W., Raida, M., Mägert, H.J., Schulz-Knappe, P. and Forssmann, W.G (1995). FEBS Lett. 368, 331-335. [6] Harder, J., Bartels, J., Christophers, E. and Schröder, J.M. (1997). Nature 387, 861. [7] Zucht, H.D., Raida, M., Adermann, K., Mägert, H.J. and Forssmann, W.G (1995). FEBS Lett. 372, 185-188. [8] Liepke, C., Zucht, H.D., Forssmann, W.G. and Ständker, L. (2001). J. Chromatogr. B 752, 369-377. [9] Yamashita, K., Kaneko, H., Yamamoto, S., Konagaya, T., Kusugami, K. and Mitsuma, T. (1998). Gastroenterology 115, 1123-1130. [10] Escher, S.E., Sticht, H., Forssmann, W.G., Rosch, P. and Adermann, K. (1999). J Pept Res. 54, 505-513. [11] Lehrer, R.I., Rosenman, M., Harwig, S.S.L., Jackson, R. and Eisenhauer, P. J. (1991). Immun. Meth. 13, 167-173. [12] Krause, A., Neitz, S., Mägert, H.-J., Schulz, A., Forssmann, W.-G., Schulz-Knappe, P. and Adermann, K. (2000) FEBS Lett. 480, 147-150. [13] Ganz, T. (1999). Science 286, 420-421. [14] Feller, S.M., Gagelmann, M., Forssmann, W.G. (1989). Trends Pharmacol. Sci. 10, 9394. [15] Kourie, J.I. (1999). FEBS Lett. 445, 57-62

[16] Kourie, J.I. (1999). Am. J. Physiol. 277, C43-C50 [17] Gerbes, A.L., Dagnino, L., Nguyen, T. and Nemer, M. (1994) J. Clin. Endocrinol. Metab. 78, 1307-1311 [18] McDonagh, T.A., Robb, S.D., Murdoch, D.R., Morton, J.J., Ford, I., Morrison, C.E., Tunstall-Pedoe, H., McMurray, J.J. and Dargie, H.J. (1998) Lancet 351, 9-13.

Figure legends Fig.1 A) Antimicrobial activity of hBNP-32 in Bacillus subtilis in comparison to other peptides and antibiotics. Upper part: size of inhibition zones (in units) A) Antimicrobial activity of hBNP32 in Bacillus subtilis in comparison to other peptides and antibiotics. Upper part: size of inhibition zones (in units) in response to hBNP-32 [g], but smaller than that induced by ampicillin [Ο]. The second messengers 8-Br-cAMP [ê] and 8-Br-cGMP [∇] (1 mM each) are inactive. Shown are the means from 4-6 repeated estimations + standard deviation of the mean. B) Image of an original agar plate carrying Bacillus subtilis and treated with increasing amounts of hBNP-32 (in µg/10µl), casocidin-1 (center; caso-1), human PTH 1-37 (PTH) or human CRH (10 µg/10 µl each) serve as negative controls.

Fig.2 Antimicrobial activity of hBNP-32 (upper part) and hCNP-22 (lower part) in S. carnosus and N. cinerea shown as the size of inhibition zones (in units). hBNP-32 is clearly more effective than hCNP-22. Shown are the means + standard deviation of n=4 representing one of three experiments.

Fig.3 Antimicrobial activity of ANP (upper part) and Urodilatin (lower part) in S. carnosus and N. cinerea shown as the size of inhibition zones (in units). ANP and Urodilatin are about equally effective. Shown are the means + standard deviation of n=4 representing one of three experiments.

Fig.4 Antimicrobial activity of hBNP-32 in S. cerevisiae shown as the size of inhibition zones (in units). HBNP-32 synthesized inhouse (A) and purchased from Sigma (B) are equally effective. Shown are the means + standard deviation of n=4 representing one of two experiments.

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