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Bactericidal activities of the cationic steroid CSA-13 and the cathelicidin peptide LL-37 against Helicobacter pylori in simulated gastric juice.

Leszczyńska K, Namiot A, Fein DE, Wen Q, Namiot Z, Savage PB, Diamond S, Janmey PA, Bucki R - BMC Microbiol. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Microbiology, Medical University of Bialystok, 15-230 Bialystok, Poland. katles@onet.eu

ABSTRACT

Background: 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.

Results: 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 microg/ml for LL-37, 17.8-142 microg/ml for WLBU2 and 0.275-8.9 microg/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.

Conclusion: 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.

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Evaluation of cell toxicity. Hemoglobin and LDH release from human red blood cells and human gastric adenocarcinoma cells (panel A and B respectively) after addition of LL-37 (circles), WLBU2 (diamonds), and CSA-13 (triangles), followed by incubation for 1 h at 37°C. Data shown are means ± SD of three experiments. Morphology of human gastric adenocarcinoma cells before (control) and after LL-37, WLBU2 and CSA-13 treatment was evaluated by phase-contrast microscopy (panel C). Data from one representative experiment are shown. Two other experiments revealed similar results.
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Figure 5: Evaluation of cell toxicity. Hemoglobin and LDH release from human red blood cells and human gastric adenocarcinoma cells (panel A and B respectively) after addition of LL-37 (circles), WLBU2 (diamonds), and CSA-13 (triangles), followed by incubation for 1 h at 37°C. Data shown are means ± SD of three experiments. Morphology of human gastric adenocarcinoma cells before (control) and after LL-37, WLBU2 and CSA-13 treatment was evaluated by phase-contrast microscopy (panel C). Data from one representative experiment are shown. Two other experiments revealed similar results.

Mentions: Non-specific insertion of antibacterial peptides and their mimics into host cell membranes can cause toxicity. Host cell membrane permeabilization can be measured by the release of proteins such as hemoglobin and LDH from the cytosol to the extracellular space. By evaluating hemoglobin and LDH release (Figure 5A and 5B), we show no significant membrane permeabilization by any tested molecules in the range at which they have bactericidal activity in saline buffers (Figure 2A, Figure 3). This finding was confirmed by microscopic evaluation of adenocarcinoma cell morphology showing no visible difference between the control cells and those treated with 10 μg/ml LL-37, WLBU2 or CSA-13 (Figure 5C). However an increase in hemoglobin and LDH release was observed with increasing concentration. Among the three molecules tested, WLBU2 was the strongest hemolytic agent, but all of them showed similar ability to compromise adenocarcinoma cell membrane integrity (Figure 5B and 5C). CSA-13 bactericidal concentrations against H. pylori and E. coli MG1655 (Figures 2A, 2B and 3C) evaluated in saline as well as nutrient containing buffer were below its minimal hemolytic concentration and below concentrations causing dysfunction of adenocarcinoma cell membranes.


Bactericidal activities of the cationic steroid CSA-13 and the cathelicidin peptide LL-37 against Helicobacter pylori in simulated gastric juice.

Leszczyńska K, Namiot A, Fein DE, Wen Q, Namiot Z, Savage PB, Diamond S, Janmey PA, Bucki R - BMC Microbiol. (2009)

Evaluation of cell toxicity. Hemoglobin and LDH release from human red blood cells and human gastric adenocarcinoma cells (panel A and B respectively) after addition of LL-37 (circles), WLBU2 (diamonds), and CSA-13 (triangles), followed by incubation for 1 h at 37°C. Data shown are means ± SD of three experiments. Morphology of human gastric adenocarcinoma cells before (control) and after LL-37, WLBU2 and CSA-13 treatment was evaluated by phase-contrast microscopy (panel C). Data from one representative experiment are shown. Two other experiments revealed similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748089&req=5

Figure 5: Evaluation of cell toxicity. Hemoglobin and LDH release from human red blood cells and human gastric adenocarcinoma cells (panel A and B respectively) after addition of LL-37 (circles), WLBU2 (diamonds), and CSA-13 (triangles), followed by incubation for 1 h at 37°C. Data shown are means ± SD of three experiments. Morphology of human gastric adenocarcinoma cells before (control) and after LL-37, WLBU2 and CSA-13 treatment was evaluated by phase-contrast microscopy (panel C). Data from one representative experiment are shown. Two other experiments revealed similar results.
Mentions: Non-specific insertion of antibacterial peptides and their mimics into host cell membranes can cause toxicity. Host cell membrane permeabilization can be measured by the release of proteins such as hemoglobin and LDH from the cytosol to the extracellular space. By evaluating hemoglobin and LDH release (Figure 5A and 5B), we show no significant membrane permeabilization by any tested molecules in the range at which they have bactericidal activity in saline buffers (Figure 2A, Figure 3). This finding was confirmed by microscopic evaluation of adenocarcinoma cell morphology showing no visible difference between the control cells and those treated with 10 μg/ml LL-37, WLBU2 or CSA-13 (Figure 5C). However an increase in hemoglobin and LDH release was observed with increasing concentration. Among the three molecules tested, WLBU2 was the strongest hemolytic agent, but all of them showed similar ability to compromise adenocarcinoma cell membrane integrity (Figure 5B and 5C). CSA-13 bactericidal concentrations against H. pylori and E. coli MG1655 (Figures 2A, 2B and 3C) evaluated in saline as well as nutrient containing buffer were below its minimal hemolytic concentration and below concentrations causing dysfunction of adenocarcinoma cell membranes.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Diagnostic Microbiology, Medical University of Bialystok, 15-230 Bialystok, Poland. katles@onet.eu

ABSTRACT

Background: 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.

Results: 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 microg/ml for LL-37, 17.8-142 microg/ml for WLBU2 and 0.275-8.9 microg/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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus