**Chromofungin**

190 Antimicrobial Agents

These new AMPs are integrated in the concept that highlights the key role of the adrenal medulla in the immunity (Sternberg, 2006) as previously reported for adrenaline and neuropeptide Y that regulate immunity systemically once released from the adrenal medulla. Furthermore, the adrenal medulla contains and releases large amounts of IL-6 and TNF-alpha in response to pro-inflammatory stimuli such as LPS, IL-1 alpha and IL-1 beta (Metz-Boutigue et al., 1998). The discovery of the presence of TLRs on the adrenal cortex cells raises the interesting possibility that the adrenal gland might have a direct role in the response to pathogens, activation of innate immune response and clearing of infectious

Several new antimicrobial peptides isolated from the granules of chromaffin cells of the bovine adrenal medulla correspond to CGA-derived peptides (Figure 3). The corresponding sequences are highly conserved in human. Interestingly, the main cleavage site in position 78-79 of bCGA and the subsequent remove of the two basic residues K77 and K78 by the carboxipeptidase H (Metz-Boutigue et al., 1993) produces two antimicrobial fragments: vasostatin-I (VS-I; bCGA1-76) (Lugardon et al., 2000) and prochromacin (Prochrom; bCGA79-431) (Strub et al.,1996b). For these N- and C-terminal domains with antimicrobial activities several shorter active fragments were identified: for VS-I, bCGA1-40 (N CgA; NCA) (Shooshtarizadeh et al., 2010), bCGA47-66 (chromofungin; CHR) and for ProChrom, bCGA173-194 (Chromacin; Chrom) (Strub et al., 1996b) and bCGA344-364 (Catestatin; CAT) (Shooshtarizadeh et al., 2010). The unique disulfide bridge of bCGA is present in VS-I and NCA sequences. Two post-translational modifications are important for the expression of the antibacterial activity of Chrom: the phosphorylation of Y173 and the O-glycosylation of S186 [130] (Strub et al., 1996a). Furthermore, it is important to point out that a dimerization motif GXXXG similar to that reported for Glycophorin A (Brosig & Langosch, 1998) is

Vasostatin-I (VS-I) displays antimicrobial activity against (i) Gram-positive bacteria (*Micrococcus luteus* and *Bacillus megaterium*) with a minimal inhibitory concentration (MIC) in the range 0.1-1 µM; (ii) against filamentous fungi (*Neurospora crassa, Aspergillus fumigatus, Alternaria brassicola, Nectria haematococca, Fusarium culmorum, Fusarium oxysporum*) with a MIC of 0.5-3 µM and (iii) against yeast cells (*Saccharomyces cerevisiae, Candida albicans*) with a MIC of 2 µM (Lugardon et al., 2000). However VS-I is unable to inhibit the growth of *Escherichia coli* SBS363 and *Escherichia coli* D22. VS-I (Figure 3) possesses structural features specific for antimicrobial peptides, such as a global positive charge (+3), an equilibrated number of polar and hydrophobic residues (20:23) and the presence of a helical region CGA40-65 characterized to be a calmodulin-binding sequence (Lugardon et al., 2001; Yoo, 1992). The loss of the antibacterial activity of CGA7-57 suggests that the N- and C-terminal sequences are essential, nevertheless CGA7-57 is less efficient than VS-I against fungi. Besides, the disulfide bridge is essential for the antibacterial, but not the antifungal property. Altogether, these data suggest that antibacterial and antifungal activities of VS-I have different structural requirements (Lugardon et al., 2001). Interestingly, two helix-helix dimerization motifs important for the interaction with membranes such as LXXXXXXL,

agents (Sternberg, 2006).

**2.2 Antimicrobial peptides derived from chromogranin A** 

present in the Chrom sequence (G184-G188).

**Vasostatin-I** 

When VS-I was treated with the endoprotease Glu-C from *Staphylococcus aureus*, one of the generated peptide, chromofungin (CHR), is the shortest active VS-I-derived peptide with antimicrobial activities (Figure 3). It is well conserved during evolution and displays antifungal activity at 2-15 µM against filamentous fungi (*Neurospora crassa, Aspergillus fumigatus, Alternaria brassicola, Nectria haematococca, Fusarium culmorum, Fusarium oxysporum*) and yeast cells (*Candida albicans, Candida tropicalis, Candida neoformans*) (Lugardon et al., 2001). Since this peptide was generated after digestion of the material present in chromaffin secretory vesicles by the endoprotease Glu-C from *S. aureus*, it may be hypothesized that it is produced during infections by this class of pathogens.

The 3-D structure of CHR has been determined in water-trifluoroethanol (50:50) by using 1H-NMR spectroscopy. This analysis revealed the amphipathic helical structure of the sequence 53-56, whereas the segment 48-52 confers hydrophobic character (Lugardon et al., 2001).The importance of the amphipathic sequence for antifungal activity was demonstrated by the loss of such activity against *N. crassa* when two proline residues were substituted for L61 and L64, disrupting the helical structure, the amphipathic character and the dimerization motif helix-helix L57-L64 (Lugardon et al., 2001).

### **Catestatin**

Two CGA-derived fragments bCGA333-364 and bCGA343-362 were characterized after the extensive processing of bCGA by prohormone convertases (PC 1/3 or 2) in chromaffin granules (Taylor et al., 2000). More recently, it was shown that cathepsin L colocalizes with CGA in chromaffin granules*. In vitro* it is able to generate after digestion of recombinant hCGA, a catestatin (CAT)–derived fragment hCGA360-373 (Biswas et al., 2009). In addition to the inhibitory effect of CAT on catecholamine release from chromaffin cells (Mahata et al., 1997), we have shown for this peptide and its shorter active sequence bCGA344-358 (cateslytin, CTL), (Figure 3) a potent antimicrobial activity with a MIC in the lowmicromolar range against Gram-positive bacteria (*Micrococcus luteus, Bacillus megaterium* at concentration of 0.8 µM), Gram-negative bacteria (*Escherichia coli D22* at concentration of 8 µM), filamentous fungi (*Neurospora crassa, Aspergillus fumigatus, Nectria haematococca* at concentration of 0.2-10 µM) and yeasts (*Candida albicans, Candida tropicalis, Candida glabrata, Candida neoformans* at concentration of 1.2-8 µM). The sequence of CAT (Figure 3) has been highly conserved during evolution (Briolat et al., 2005). The two human variants P370L and

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

bacteria.

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 193

of bacterial proteases on the isolated AMPs derived from CGs, we have tested the effects of *Staphylococcus aureus* V8 protease Glu-C and several supernatants from *S aureus*, *Salmonella enteretica*, *Klebsiella oxytoca*, *Shigella sonnei* and *Vibrio cholera*. By using biochemical methods we have analyzed the degradation of the peptide in presence of

*Interaction of antimicrobial CGs-derived peptides with proteases from diarrheogenic bacteria* Bacteria were isolated from patients of the Strasbourg Civil Hospital by the Bacteriology Institute, University of Strasbourg, (EA-4438). The four strains have a clinical interest

Thus, *Klebsiella* was involved in the occurrence of post-antibiotic diarrheas (Gorkiewicz 2009). Many studies show that *Klebsiella oxytoca* is also involved in nosocomial infections for newborns or adults (Biran et al., 2010) *Klebsiella* infections may also be commensal (Tsakris et al., 2011). *Klebsiella oxytoca* has also been associated with hemorrhagic colitis (Hoffmann et al., 2010) and intercurrent colitis in Crohn's disease (Plessier et al. 2002). *Salmonella* destroys infected cells and the infection continues through blood (sepsis) or through lymphatic vessel (typhoid fever). Salmonella cause also gastrointestinal infections. *Shigella sonnei* and *Vibrio cholera* non O1 cause inflammation of the intestinal

*Klebsiella oxytoca, Salmonella enterica, Shigella sonnei, and Vibrio cholera* develop phenomena of antibiotic resistance. Thus, *Salmonella* was reported to be resistant for the action of

Concerning CgA, we have tested bovine, rat and human CAT corresponding to the sequences bCgA344-364, rCgA6344-364 and hCgA352-372, bovine CTL located at bCgA344- 358, two short fragments hCgA360-372 and the conserved tetrapeptide LSFR (bCgA348-351). In addition, we have tested a scrambled peptide relative to the sequence of bovine CAT and

We have found antimicrobial activities only for the bovine CAT and CTL, showing that CTL is the shorter active fragment and that it corresponds to the active domain of CAT. Procatestatin was inactive in similar experimental conditions. Bovine CAT and CTL were active against *Klebsiella oxytoca*, *Salmonella enterica* and *Vibrio cholera* at 100 µM and 50 µM respectively and against *Shigella Sonnei* at 50 µM and 25 µM. In addition, CHR and the Cterminal fragment (CgA387-431) were inactive for concentration up to 100 µM. In contrast,

Three CgB-derived peptides (CgB58-62, CgB279-291, and CgB547-560) and secretoneurin corresponding to SgII189-254 were examined against the four strains in order to analyse their degradation by bacterial proteases. By using HPLC we have compared the profiles of

These experiments show that except CTL all the peptides are completely degraded by the bacteria. To illustrate these data, we present on Figure 4, the profiles relative to CAT and CTL in presence of buffer with *Salmonella enterica*. The complete peptide and the processed

because apart from inducing diarrhea, they may cause other infections.

Ciprofloxacine (Medalla et al., 2011) and Ceftriaxone (Su et al., 2011).

mucosa by producing the Shiga toxin.

the procatestatin fragment bCgA332-364.

CTL is active at 30 µM against the four pathogens.

the peptide alone and the peptide with the inoculated medium.

form are analysed by sequencing and mass spectrometry (MALDI-TOF).

G364S display antibacterial activity against *M. luteus* with a MIC of 2 and 1 µM, respectively, and against *E. coli* with a MIC of 20 and 10 µM, respectively (Briolat et al., 2005). However, the most active peptide corresponds to the bovine sequence. Bovine CTL, a cationic sequence with a global net charge of +5 (R344, R347, R351, R353, R358) and five hydrophobic residues (M346, L348, F360, Y355, F357) (Figure 3), is able to completely kill bacteria at concentration lower than 10 µM even in the presence of NaCl (0-150 mM) (Briolat et al., 2005). The C-terminal sequence bCGA352-358 is inactive, whereas the N-terminal sequences bCGA344-351 and bCGA 348-358 are antibacterial at 20 µM.
