Bactericidal activities of the cationic steroid CSA-13 and the cathelicidin peptide LL-37 against Helicobacter pylori in simulated gastric juice
© Leszczyńska et al; licensee BioMed Central Ltd. 2009
Received: 28 April 2009
Accepted: 3 September 2009
Published: 3 September 2009
The worldwide appearance of drug-resistant strains of H. pylori motivates a search for new agents with therapeutic potential against this family of bacteria that colonizes the stomach, and is associated with adenocarcinoma development. This study was designed to assess in vitro the anti-H. pylori potential of cathelicidin LL-37 peptide, which is naturally present in gastric juice, its optimized synthetic analog WLBU2, and the non-peptide antibacterial agent ceragenin CSA-13.
In agreement with previous studies, increased expression of hCAP-18/LL-37 was observed in gastric mucosa obtained from H. pylori infected subjects. MBC (minimum bactericidal concentration) values determined in nutrient-containing media range from 100-800 μg/ml for LL-37, 17.8-142 μg/ml for WLBU2 and 0.275-8.9 μg/ml for ceragenin CSA-13. These data indicate substantial, but widely differing antibacterial activities against clinical isolates of H. pylori. After incubation in simulated gastric juice (low pH with presence of pepsin) CSA-13, but not LL-37 or WLBU2, retained antibacterial activity. Compared to LL-37 and WLBU2 peptides, CSA-13 activity was also more resistant to inhibition by isolated host gastric mucins.
These data indicate that cholic acid-based antimicrobial agents such as CSA-13 resist proteolytic degradation and inhibition by mucin and have potential for treatment of H. pylori infections, including those caused by the clarithromycin and/or metronidazole-resistant strains.
Helicobacter pylori is carried by more than half of the world's adult population . It can chronically colonize the human gastric mucosa, where it is found in the mucus layer and is adhered to epithelial cells . Although most infected subjects remain asymptomatic, infection with H. pylori can promote severe gastritis  and significantly increase the risk of gastric malignancies [4, 5]. In some epidemiological studies, H. pylori eradication was shown to be effective in gastric cancer prevention [6, 7]. Additionally, H. pylori eradication was found to decrease the incidence and the severity of lesions with carcinogenic potential in animal models [8, 9]. Natural mechanisms that protect the host from H. pylori infections depend on the function of the innate defense system in which antibacterial peptides such as cathelicidin LL-37 [10, 11] and O-glycans in gastric mucin  play a key role.
LL-37 is a proteolytically processed peptide derived from the C-terminal domain of human cathelicidin (hCAP-18/LL-37) that is constitutively released to the extracellular space by phagocytic granulocytes and epithelial cells . Functions ascribed to LL-37 include prevention of bacterial growth , neutralization of bacterial wall molecule bioactivity , and activation of host cells by binding specific cell membrane receptors [16–18]. H. pylori upregulates the production of LL-37/hCAP18 by the gastric epithelium, suggesting that cathelicidin or its derivative LL-37 contributes to determining the balance between host mucosal defense and H. pylori survival mechanisms that govern chronic infection with this gastric pathogen [10, 11].
Cationic antibacterial peptides (CAPs) including LL-37 have been extensively investigated as a potential source of new antibacterial molecules. The engineered WLBU2 peptide whose residues are arranged to form an amphipathic helical structure with optimal charge and hydrophobic density, overcomes some limitations of natural LL-37 such as sensitivity to Mg2+ or Ca2+ and inactivation by blood serum . Therefore WLBU2 could treat infections where LL-37 is ineffective. In order to generate molecules able to mimic CAPs' ability to compromise bacterial membrane integrity, non-peptide ceragenins with cationic, facially amphiphilic structures characteristic of most antimicrobial peptides were developed. Ceragenins such as CSA-13 reproduce the required CAP morphology using a bile-acid scaffolding and appended amine groups . They are bactericidal against both Gram-positive and Gram-negative organisms, including drug-resistant bacteria such as clinically relevant methicillin-resistant Staphylococcus aureus (MRSA), and a previous susceptibility study demonstrated that CSA-13 has a MIC50/MBC50 ratio of 1 [21, 22]. In this study we compare the bactericidal potency of LL-37, WLBU2 and CSA-13 against clinical isolates of H. pylori. The results suggest that cholic acid-based mimics of antimicrobial peptide such as CSA-13 have potential for treatment of H. pylori infection, including those caused by the clarithromycin and/or metronidazole-resistant strains.
Immunohistochemical probing of human gastric mucosa sections with anti-hCAP-18/LL-37 antibody
Bactericidal activity of LL-37, WLBU-2 peptides and ceragenin CSA-13 against different strains of H. pylori
Evaluation of sensitivity of clinical strains of H. pylori to antibiotics.
H. pylori strains
Antibacterial activity of LL-37, WLBU2 and CSA-13 after pre-incubation at low pH with pepsin or mucin
Analytical characterization of LL-37 and CSA-13 after incubation with pepsin
Toxicity of LL-37, WLBU2 and CSA-13 against RBC and human adenocarcinoma cells
The rate of successful treatment of H. pylori stomach infection, achieved with combination therapies of two antibiotics and a proton pump inhibitor has declined from over 90% to about 80% during the past decade . In addition, the cost of this therapy is significant, and therefore a need for more widely available means of treating or preventing H. pylori infection still exists . New agents to treat H. pylori infections are necessary also due to increasing drug-resistance problems caused by extensive use of antibiotics  and the adaptive survival mechanisms of pathogenic bacteria to counteract currently used antimicrobials. For example, H. pylori strains resistant to amoxicillin, metronidazole and clarithromycin have been reported [30, 31]. Methods to improve treatments for H. pylori might be guided by insight into the natural mechanisms by which infected patients respond to this bacterium and the reasons why the normal host-defense mechanisms fail.
This study confirms a previous report of increased hCAP-18/LL-37 expression in gastric mucosa of subjects infected with H. pylori . This finding suggests that increasing production of the bactericidal peptide LL-37 may play a key role in host defense against H. pylori . However, this bactericidal response in some subjects is insufficient and H. pylori infection can still reach a chronic stage. The lack of bactericidal function of LL-37 in this setting has suggested that increased expression of hCAP-18/LL-37 peptide in gastric mucus of infected subjects may have additional functions as an anti-inflammatory and growth stimulating agent. Indeed, it was recently shown that gastric ulcer healing in rats is promoted by cathelicidin-mediated transactivation of epidermal growth factor receptors (EGFR) via the transforming growth factor alpha (TGFα) signaling pathway . Alternatively, loss of defense against H. pylori may be due to loss of antibacterial function of LL-37 in the milieu of the gastric mucosa. Consequently, design of antimicrobial agents that are more effective in this setting can be beneficial.
Motivated by immunohistological results, the activity of LL-37 against clinical isolates of H. pylori and E. coli MG1655 under biologically relevant conditions was compared with that of the synthetic peptide WLBU2 and the ceragenin CSA-13. This study shows that CSA-13, contrary to LL-37 and WLBU2 peptides, maintains strong bactericidal activity in the presence of mucin and after preincubation with pepsin at low pH. These conditions represent unique challenges related to H. pylori treatment, as these bacteria in the stomach are protected from the acidic environment by a thick mucus layer and the effectiveness of many antimicrobial drugs is greatly diminished at acidic pH . Accordingly, the first effective therapy for H. pylori infection was a combination of relatively pH-insensitive antimicrobial drugs such as bismuth, tetracycline and metronidazole . In addition, as the stomach periodically empties its contents (topical therapy tends to be diluted and washed out) the finding that CSA-13 has bactericidal activity at much lower concentration then LL-37, after the same incubation time (3-6 hours) , suggests that CSA-13 may have therapeutic potential for treatment of H. pylori infection. The antibacterial activity of CSA-13, which has a smaller net charge and a unique distribution of this charge over a steroid scaffold when compared with LL-37 and WLBU2 peptides, was also found to be less inhibited by mucin isolated from gastric mucosa. Therapeutic potential based on the ability of CSA-13 to eradicate H. pylori is also supported by previously reported antibacterial activity against other bacteria strains, including clinical isolates of Pseudomonas aeruginosa  and S. aureus . CSA-13's unique ability to compromise bacterial membrane integrity and the chemical nature of this low-molecular-mass compound that translates to lower cost of synthesis compared to cationic antibacterial peptides suggest that CSA-13 or perhaps other ceragenins have potential for treatment of H. pylori infection, including those caused by its resistant strains.
Bactericidal activity of ceragenin CSA-13 is maintained after preincubation in simulated gastric juice and in the presence of mucin. This in vitro evaluation indicates a significant potential of this molecule in treatment of stomach mucosal infection.
LL-37 (NH2-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-COOH) and WLBU2 (NH2-RRWVRRVRRWVRRVVRVVRRWVRR-COOH) peptides were purchased from Bachem (King of Prussia, PA). CSA-13 was prepared as previously described . Amoxicillin (AMX), clarithromycin (CLR), tetracycline (TET) and metronidazole were purchased from Sigma.
Collection of gastric mucosal and bile samples
During gastroscopy, performed with either a GIF V2 or Q145 (Olympus) gastroscope, several gastric mucosal slices were taken from the prepyloric and corpus regions of the stomach. H. pylori infection was established in the biopsy specimens using a urease test (CLO-test). Human bile was obtained from the gallbladder of patients undergoing cholecystectomy. Samples were filter-sterilized through a 0.45 μm membrane before being diluted in PBS (1:1) and mixed with antibacterial agents used in bacteria killing assays. The studies were approved by Medical University of Bialystok Ethics Committee for Research on Humans and Animals, and all patients gave informed written consent for participation in the study.
Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded human gastric mucosal sections using a rabbit anti-LL-37 antibody (H-075-06, used at 1:100 dilution; Phoenix Pharamceuticals Inc.). Paraffin-embedded materials were sectioned to 5 μm thickness and floated on distilled water at 45°C. Sections were then mounted on slides and placed in 57°C oven overnight. The sections were deparaffinized according to standard procedures and quenched with 0.9% hydrogen peroxide in methanol for 30 minutes. The sections were incubated with primary antibody at 37°C for 60 minutes, washed with 1% PBSA (1% BSA in PBS), and subjected to binding with secondary antibody (biotinylated goat anti-Rabbit IgG, 1:400 dilution). Amplification was performed with a Vectastain ABC kit, and an HRP detection system was used to colocalize peroxidase activity with a DAB substrate. The sections were counterstained with hematoxylin. Samples were viewed with a Nikon Eclipse 80 microscope under 40× magnification.
Evaluation of MIC and MBC
The minimal inhibitory concentration (MIC) of conventional antibiotics against seven different clinical isolates of H. pylori (9 × 108 CFU/ml) was determined using Muller-Hinton agar (MH) containing 5% sheep blood. The incubation was continued for 4 days at 35°C in microaerophilic conditions maintained with use of a Gas Pack-Campylobacter gas generating kit BR60. Clinical isolates of H. pylori were considered resistant to respective antibiotics when the MIC values were above 4 μg/ml for AMX, 1 μg/ml for CLR and 16 μg/ml for TET and Metronidazole. The minimal bactericidal concentration (MBC) of antibacterial agents was evaluated using an inoculum at 108 CFU/ml. After a 6-hour incubation at 37°C, 10 μl aliquots of the suspensions were spotted on Columbia agar supplemented with sheep blood (5%).
Bacteria killing assay
The bactericidal activities of LL-37, WLBU2 peptides and ceragenin CSA-13 against E. coli MG1655 in the presence of mucin or pepsin from porcine mucus (Sigma) and human bile were measured as previously described . Bacteria were grown to mid-log phase at 37°C (controlled by the evaluation of optical density at 600 nm) and resuspended in PBS buffer (pH = 7.4). The bacteria suspensions were then diluted 10 times in 100 μl of solutions containing antibacterial agents by themselves or with mucin (1000 μg/ml), or bile (the final 1:10 bile dilution mimics the environment of the upper small intestine into which bile is secreted  (pH = 7.4)). In another set of experiments antibacterial activity of these components was determined following their preincubation in simulated gastric juice [36, 37] at pH ~1.5 with and without pepsin (0.5 mg/ml). After incubating bacteria with antibacterial molecules for one-hour at 37°C, the bacterial suspensions were placed on ice and diluted 10- to 1000- fold. Aliquots of each dilution (10 μl) were spotted on LB Agar plates for overnight culture at 37°C. The number of colonies at each dilution was counted the following morning. The colony forming units (CFU/ml) of the individual samples were determined from the dilution factor.
Analytical characterization was performed on the CSA-13 and LL-37 suspensions after 3H incubation with pepsin (0.5 mg/ml) at low pH (~1,5) at 37°C, using the Shimadzu (Columbia, MD) instrument (the LC-MS system consisted of a LC-20AB solvent delivery system and SIL-20A auto-sampler coupled to dual wavelength UV-Vis detector and a LCMS 2010EV single quadrupole mass spectrometer), coupled to a Shimadzu Premier C18 column (150 mm × 4.6 mm i.d., 5 μm particle size). The mobile phase flow rate was 1 ml/min with a starting ratio of 90% mobile phase A (water) and 10% mobile phase B (acetonitrile) both with 0.1% (v/v) formic acid. The analytical method consisted of the following steps: (i) sample injection and holding at 10% B for 5 min, (ii) linear gradient from 10% to 90% B over 15 minutes, (iii) holding at 90% B for 5 minutes, (iv) isocratic step to 10% B and holding for 5 minutes prior to the next sample injection. Mass spectrometry was performed on the eluent using electrospray ionization (ESI) in positive ion mode with a scanned m/z range from 160-2000.
Red blood cell lysis
The hemolytic activity of LL-37, WLBU-2 and CSA-13 (0-200 μ g/ml), against human red blood cells (RBC) was tested using erythrocytes suspended in PBS. RBC prepared from fresh blood (Hematocrit ~5%) were incubated for 1 h at 37°C after addition of test molecules. Relative hemoglobin concentration in supernatants after centrifugation at 2000 × g was monitored by measuring the absorbance at 540 nm. 100% hemolysis was taken from samples in which 2% Triton X-100 was added.
Human gastric adenocarcinoma cells (ATCC; CRL-1739) were maintained in DMEM (BioWhittaker) culture supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) at 37°C and 5% CO2. For LDH release assay and microscope evaluation cells were plated in 24 well plates and grown to confluence. In all experiments, the medium was changed to serum-free media ~12 h prior to cell treatment with LL-37, WLBU2 and CSA-13 (0-200 μg/ml) in individual wells, for 1 hour. Cell culture medium was then collected, centrifuged (10 mins, 5000 rpm, RT) and subjected to LDH evaluation (LDH-cytotoxicity Assay Kit; BioVision Inc.)
This work was supported by NIH grant HL067286 and Medical University of Bialystok grants 3-22458F and 3-18714L
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