**3. Interaction of antimicrobial chromogranins-derived peptides with bacterial proteases**

The AMPs avoidance mechanisms deployed by bacteria include the proteolytic degradation of the active forms by the bacterial proteases. In order to examine the effects

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

CCA, the C-terminal CGA-derived fragment bCGA418-427 (Figure 3), with a remarkable net charge of -2, displays antifungal activity and belongs to the less abundant anionic AMPs family. It is well conserved during evolution and is homologous to the C-terminal sequence of CGB and the antibacterial peptide SEC (secretolytin) [(Strub et al., 1996b). This peptide was generated *in vitro* after digestion, by the protease Glu-C from *S. aureus*, of the material present in chromaffin secretory vesicles. As previously postulated for CHR, CCA could be

To date, the natural C-terminal fragment of bovine CGB (CCB; bCGB 564-626), isolated from chromaffin granules of the adrenal medulla, was found to display antibacterial activity against both *M. luteus* and *E. coli*. The complete inhibition of bacterial growth was observed at a concentration around 1.8 µM (Strub et al., 1996b). This large fragment contains the natural short antibacterial peptide secretolytin (SEC, bCGB614-626) with a net positive charge (+3) (Figure 3). We observed the natural formation of a pyrrolidone glutamic acid at the N-terminal end of SEC and both forms displayed antibacterial activity against *M. luteus*, reaching 100% of growth inhibition at 2 µM (Strub et al., 1996ab). A structure-activity analysis suggests that an alpha-helical amphipathic structure common to SEC and cecropins

Because bSGII is weakly expressed in the intragranular matrix of chromaffin secretory vesicles (2% of total proteins), the detection of endogenous AMPs by classical methods was unsuccessful. After *in-silico* analysis, two synthetic peptides with cationic amphipathic sequences were prepared: Rrf and Kvk, which correspond to the sequences bSGII131-138 and bSGII430-443 with respective net charges of +4.5 and 4 (Figure 3). Rrf, completely inhibits the bacterial growth of *M. luteus* and *B. megaterium* with a MIC of 5 and 15 µM, respectively, and Kvk displays antifungal properties at 19 µM against *N. crassa*

**3. Interaction of antimicrobial chromogranins-derived peptides with bacterial** 

The AMPs avoidance mechanisms deployed by bacteria include the proteolytic degradation of the active forms by the bacterial proteases. In order to examine the effects

sequences bCGA344-351 and bCGA 348-358 are antibacterial at 20 µM.

**2.3 Antimicrobial peptides derived from bovine chromogranin B** 

may account for the antibacterial activity (Strub et al., 1996b).

**2.4 Antimicrobial peptides derived from bovine secretogranin II** 

generated during infections induced by this pathogen.

**C-terminal CGA-derived fragment** 

(Shooshtarizadeh et al., 2010).

**proteases** 

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 bacteria.

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

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 mucosa by producing the Shiga toxin.

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

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 the procatestatin fragment bCgA332-364.

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, CTL is active at 30 µM against the four pathogens.

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 the peptide alone and the peptide with the inoculated medium.

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

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

min−1 using the gradient indicated on the chromatogram.

represent interesting candidates to synergistically act with antibiotics.

**4.1 For** *Staphylococcus aureus* **MRSA** 

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 195

Fig. 5. Analysis by HPLC of the catestatin (CAT) degradation by a methicillin resistant *Staphylococcus aureus* (MRSA). The HPLC system is composed by a Dionex chromatograph, (Germerong, Germany), using a Nucleosil 300-5 C18 column (4×250 mm, particle size 5 μm, porosity 300 Å; Macherey Nagel, Düren, Germany). The solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.09% (v/v) trifluoroacetic acid in 70% acetonitrile in milliQ water (solvent B). Elutions were performed at a flow rate of 700 μL

**4. Synergy of the combination of antimicrobial peptides with antibiotics** 

The emergence of multi-drug resistant bacteria (MDR), with therapeutic failure against *Staphylococcus aureus* (MRSA), *Klebsiella pneumonia*, *Acinebacter baumannii and Pseudomonas aeruginosa* have paved the way to develop new therapeutic agents by the help of the synergism. In addition the highly toxic effects of antibiotics have shifted the research focus to discover new peptides with broad spectrum of activity and less toxicity. Synergy is the combined activity of two antimicrobial agents that can never be attained by any one of them singly (Serra et al., 1977). Numerous AMPs demonstrate broad spectrum of activity against pathogens, interacting directly with membranes or acting with a specific mode. They

Most of the patients can prone to serious bacterial infections caused mainly by the multiresistant microorganisms, *Staphylococcus aureus* coagulase negative spp are one of them. These coagulase negative strains (MRSA) have got approximately 90% of methicillin resistance due to β-lactam resistance (Silva et al., 2011). Story just not stopped here, but still it continues, some of the staphylococcal strains got resistance to the other drugs such as Vancomycin, which was previously widely used against the MRSA infections, and to treat

Fig. 4. Analysis by HPLC of the catestatin (CAT) and cateslytin (CTL) degradation by *Salmonella enterica.* The HPLC system is composed by a Dionex chromatogram, Germerong, Germany), using a Nucleosil 300-5 C18 column (4×250 mm, particle size 5 μm, porosity 300 Å; Macherey Nagel, Düren, Germany). The solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.09% (v/v) trifluoroacetic acid in 70% acetonitrile in milliQ water (solvent B). Elutions were performed at a flow rate of 700 μL min−1 using the gradient indicated on the chromatogram.

#### **3.1 Interaction of antimicrobial Cgs-derived peptides with proteases from**  *Staphylococcus aureus*

After incubation with *S. aureus* V8 protease Glu-C of the proteic intragranular material of chromaffin cells present in the adrenal medulla, 21 new peptides were isolated by HPLC and analysed by sequencing and mass spectrometry. These peptides were tested against Gram positive bacteria (*Micrococcus luteus* and *S. aureus*), Gram negative bacteria (*Escherichia coli*), fungi (*Neurospora crassa*) and yeast (*Candida albicans*). They are not antibacterial but 5 peptides corresponding to CgA47-60, CgA418-426 and CgB 279-291, CgB 450-464 and CgB470-486 display antifungal activity at the micromolar range against *N. crassa*. Thus, *S. aureus* subverts innate immunity to degrade the antibacterial Cgs/Sgs-derived peptides and produce new antifungal peptides (manuscript in preparation).

Four antimicrobial CgA derived peptides (CHR CgA47-66, bovine CAT CgA344-364, human CgA352-372 and CTL CgA344-358) were incubated in presence of staphylococcal supernatants from S1 (a Methicillin resistant strain) and S2 (a non-resistant strain). CTL, the active domain of CAT, is able to completely kill *S. aureus* at 30 µM, but the two others peptides are inactive. By using a proteomic analysis (HPLC, sequencing and mass spectrometry) we demonstrated that CHR and CTL were not degraded by supernatants, whereas bovine and human CAT are processed to produce several fragments (Figure 5).

Fig. 4. Analysis by HPLC of the catestatin (CAT) and cateslytin (CTL) degradation by *Salmonella enterica.* The HPLC system is composed by a Dionex chromatogram, Germerong, Germany), using a Nucleosil 300-5 C18 column (4×250 mm, particle size 5 μm, porosity 300

Å; Macherey Nagel, Düren, Germany). The solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.09% (v/v) trifluoroacetic acid in 70% acetonitrile in milliQ water (solvent B). Elutions were performed at a flow rate of 700 μL

**3.1 Interaction of antimicrobial Cgs-derived peptides with proteases from** 

After incubation with *S. aureus* V8 protease Glu-C of the proteic intragranular material of chromaffin cells present in the adrenal medulla, 21 new peptides were isolated by HPLC and analysed by sequencing and mass spectrometry. These peptides were tested against Gram positive bacteria (*Micrococcus luteus* and *S. aureus*), Gram negative bacteria (*Escherichia coli*), fungi (*Neurospora crassa*) and yeast (*Candida albicans*). They are not antibacterial but 5 peptides corresponding to CgA47-60, CgA418-426 and CgB 279-291, CgB 450-464 and CgB470-486 display antifungal activity at the micromolar range against *N. crassa*. Thus, *S. aureus* subverts innate immunity to degrade the antibacterial Cgs/Sgs-derived peptides and

Four antimicrobial CgA derived peptides (CHR CgA47-66, bovine CAT CgA344-364, human CgA352-372 and CTL CgA344-358) were incubated in presence of staphylococcal supernatants from S1 (a Methicillin resistant strain) and S2 (a non-resistant strain). CTL, the active domain of CAT, is able to completely kill *S. aureus* at 30 µM, but the two others peptides are inactive. By using a proteomic analysis (HPLC, sequencing and mass spectrometry) we demonstrated that CHR and CTL were not degraded by supernatants, whereas bovine and human CAT are processed to produce several fragments (Figure 5).

min−1 using the gradient indicated on the chromatogram.

produce new antifungal peptides (manuscript in preparation).

*Staphylococcus aureus* 

Fig. 5. Analysis by HPLC of the catestatin (CAT) degradation by a methicillin resistant *Staphylococcus aureus* (MRSA). The HPLC system is composed by a Dionex chromatograph, (Germerong, Germany), using a Nucleosil 300-5 C18 column (4×250 mm, particle size 5 μm, porosity 300 Å; Macherey Nagel, Düren, Germany). The solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (solvent A) and 0.09% (v/v) trifluoroacetic acid in 70% acetonitrile in milliQ water (solvent B). Elutions were performed at a flow rate of 700 μL min−1 using the gradient indicated on the chromatogram.
