Research Article

Interaction with lipopolysaccharide is key to efficacy of tryptophan- and arginine-rich α-melanocyte-stimulating hormone analogs against Gram-negative bacteria

Antimicrobial Research Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

‡Authors contributed equally

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Priya Patel‡

Antimicrobial Research Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

‡Authors contributed equally

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Aftab H Mondal

Antimicrobial Research Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

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Kasturi Mukhopadhyay

*Author for correspondence:

E-mail Address: kasturim@mail.jnu.ac.in

https://orcid.org/0000-0002-6886-1080

Antimicrobial Research Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

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Published Online:21 Dec 2023https://doi.org/10.2217/fmb-2023-0080

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Abstract

Aim: In order to search for novel antibacterial therapeutics against Gram-negative bacteria, the antibacterial efficacies and mechanism of action of tryptophan- and arginine-rich α-melanocyte-stimulating hormone analogs were investigated. Materials & methods: We performed a killing assay to determine their efficacy; fluorescence, microscopic studies were used to understand their mechanism and peptide–lipopolysaccharide interaction. A checkerboard assay was used to find the effective combination of peptide and antibiotics. Results: Ana-peptides displayed good killing activity against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Their strong interaction with lipopolysaccharide damaged the bacterial membranes and led to their subsequent death. Ana-5, the highest cationic and hydrophobic analog, emerged as the most potent peptide, showing synergistic action with rifampicin and erythromycin. Conclusion: Ana-5 can be presented as an important therapeutic candidate against bacterial infections.

Plain language summary

Bacteria can cause infections. These infections are becoming harder to treat, because excessive use of antibiotics can cause these bacteria to become less susceptible to medicine. In hospitals, these bacteria can cause infections in the lungs, urinary tract, blood, or on the skin. Our bodies make small molecules called antimicrobial peptides (AMPs) to fight against bacteria. AMPs can weaken or quickly destroy bacteria by attaching to their surfaces and breaking them down. Our laboratory has made an AMP called Ana-5. Using Ana-5 with regular medicine is better at killing bacteria. Ana-5 is not only good at fighting these bacteria, but may also help to prevent future infections.

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References

1. Daoud Z, Dropa M. Editorial: the global threat of carbapenem-resistant Gram-negative bacteria. Front. Cell. Infect. Microbiol. 2(13), 1196488 (2023). •• Emerging resistance in Gram-negative bacteria against the conventional antibiotics.
Google Scholar
2. Asokan GV, Ramadhan T, Ahmed E, Sanad H. WHO global priority pathogens list: a bibliometric analysis of Medline–PubMed for knowledge mobilization to infection prevention and control practices in Bahrain. Oman Med. J. 34(3), 184–193 (2019).
Medline CASGoogle Scholar
3. Centers for Disease Control and Prevention. Antibiotic resistance threats in the United States, 2019 (2019). www.cdc.gov/DrugResistance/Biggest-Threats.html
Google Scholar
4. Mogasale VV, Saldanha P, Pai V, Rekha PD, Mogasale V. A descriptive analysis of antimicrobial resistance patterns of WHO priority pathogens isolated in children from a tertiary care hospital in India. Sci. Rep. 11, 5116 (2021).
Medline CASGoogle Scholar
5. Tacconelli E, Carrara E, Savoldi A, Kattula D, Burkert F. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. Lancet Infect. Dis. 18(3), 318–327 (2018).
MedlineGoogle Scholar
6. Santos MA, Silva FL, Lira BOV et al. Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for Gram-negative bacteria. Biochim. Biophys Acta Gen. Subj. 1867(1), 130265 (2023).
Medline CASGoogle Scholar
7. van Duin D, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae. Virulence 8(4), 460–469 (2017).
Medline CASGoogle Scholar
8. Magiorakos A-P, Srinivasan A, Carey RB et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18(3), 268–281 (2012).
Medline CASGoogle Scholar
9. Breijyeh Z, Jubeh B, Karaman R. Resistance of Gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules 25(6), 1340 (2020).
Medline CASGoogle Scholar
10. Ghai I, Ghai S. Exploring bacterial outer membrane barrier to combat bad bugs. Infect. Drug Resist. 10, 261–273 (2017).
Medline CASGoogle Scholar
11. Bertani B, Ruiz N. Function and biogenesis of lipopolysaccharides. EcoSal Plus 8(1), doi: 10.1128/ecosalplus.ESP-0001-2018 (2018).
MedlineGoogle Scholar
12. Poirel L, Madec J-Y, Lupo A et al. Antimicrobial resistance in Escherichia coli. Microbiol. Spectr. 6(4), ARBA-0026-2017 (2018).
Google Scholar
13. Mwangi J, Hao X, Lai R, Zhang Z-Y. Antimicrobial peptides: new hope in the war against multidrug resistance. Zool. Res. 40(6), 488–505 (2019).
Medline CASGoogle Scholar
14. Barreto-Santamaría A, Arévalo-Pinzón G, Patarroyo MA, Patarroyo ME. How to combat Gram-negative bacteria using antimicrobial peptides: a challenge or an unattainable goal? Antibiotics 10(12), 1499 (2021). •• A detailed description of antimicrobial peptides, their structure, stability, use against resistant bacteria and limitations.
CASGoogle Scholar
15. Huan Y, Kong Q, Mou H, Yi H. Antimicrobial peptides: classification, design, application and research progress in multiple fields. Front. Microbiol. 11, 2559 (2020).
Google Scholar
16. Soundrarajan N, Park S, Le Van Chanh Q et al. Protegrin-1 cytotoxicity towards mammalian cells positively correlates with the magnitude of conformational changes of the unfolded form upon cell interaction. Sci. Rep. 9, 11569 (2019).
MedlineGoogle Scholar
17. Kouno T, Fujitani N, Mizuguchi M et al. A novel β-defensin structure: a potential strategy of big defensin for overcoming resistance by Gram-positive bacteria. Biochemistry 47(40), 10611–10619 (2008). •• Importance of α-melanocyte-stimulating hormone-based peptides as emerging host defense peptides, their stability and broad-spectrum activity.
Medline CASGoogle Scholar
18. Singh M, Mukhopadhyay K. Alpha-melanocyte stimulating hormone: an emerging anti-inflammatory antimicrobial peptide. Biomed. Res. Int. 2014, 874610 (2014).
MedlineGoogle Scholar
19. Singh M, Mukhopadhyay K. C-terminal amino acids of alpha-melanocyte-stimulating hormone are requisite for its antibacterial activity against Staphylococcus aureus. Antimicrob. Agents Chemother. 55(5), 1920–1929 (2011).
Medline CASGoogle Scholar
20. Tiwari K, Singh M, Kumar P, Mukhopadhyay K. Binding of cationic analogues of α-MSH to lipopolysaccharide and disruption of the cytoplasmic membranes caused bactericidal action against Escherichia coli. Sci. Rep. 12, 1987 (2022).
Medline CASGoogle Scholar
21. Singh J, Joshi S, Mumtaz S et al. Enhanced cationic charge is a key factor in promoting staphylocidal activity of α-melanocyte stimulating hormone via selective lipid affinity. Sci. Rep. 6, 31492 (2016).
Medline CASGoogle Scholar
22. Shireen T, Singh M, Dhawan B, Mukhopadhyay K. Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone. Peptides 37, 334–339 (2012).
Medline CASGoogle Scholar
23. Singh J, Mumtaz S, Joshi S, Mukhopadhyay K. In vitro and ex vivo efficacy of novel Trp–Arg rich analogue of α-MSH against Staphylococcus aureus. ACS Omega 5(7), 3258–3270 (2020). •• Description of the various methods used for determining the efficacy and mechanism of action of antimicrobial peptides against pathogenic bacteria.
Medline CASGoogle Scholar
24. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial testing (CLSI supplement M100). 31st Edition. Clinical and Laboratory Standards Institute, PA, USA (2021).
Google Scholar
25. Dong N, Wang C, Zhang T et al. Bioactivity and bactericidal mechanism of histidine-rich β-hairpin peptide against Gram-negative bacteria. Int. J. Mol. Sci. 20(16), 3954 (2019).
Medline CASGoogle Scholar
26. Wang J, Song J, Yang Z et al. Antimicrobial peptides with high proteolytic resistance for combating Gram-negative bacteria. J. Med. Chem. 62(5), 2286–2304 (2019).
Medline CASGoogle Scholar
27. Saravanan R, Holdbrook DA, Petrlova J et al. Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides. Nat. Commun. 9(1), 2762 (2018).
MedlineGoogle Scholar
28. Fernandez RM, Ito AS, Schiöth HB, Lamy MT. Structural study of melanocortin peptides by fluorescence spectroscopy: identification of β-(2-naphthyl)-D-alanine as a fluorescent probe. Biochim. Biophys Acta Gen. Subj. 1623(1), 13–20 (2003).
CASGoogle Scholar
29. Petrlova J, Petruk G, Huber RG et al. Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products. J. Biol. Chem. 295(11), 3417–3430 (2020).
Medline CASGoogle Scholar
30. Yasir M, Dutta D, Willcox MDP. Comparative mode of action of the antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa. Sci. Rep. 9, 7063 (2019).
MedlineGoogle Scholar
31. Datta A, Kundu P, Bhunia A. Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: structural insights into lipopolysaccharide binding. J. Colloid Interface Sci. 461, 335–345 (2016).
Medline CASGoogle Scholar
32. Zhu N, Zhong C, Liu T et al. Newly designed antimicrobial peptides with potent bioactivity and enhanced cell selectivity prevent and reverse rifampin resistance in Gram-negative bacteria. Eur. J. Pharm. Sci. 158, 105665 (2021).
Medline CASGoogle Scholar
33. Mumtaz S, Behera S, Joshi S, Mukhopadhyay K. Efficacy and toxicity studies of novel α-MSH analogues with antibiofilm action and β-lactam resensitization potential against MRSA. ACS Infect. Dis. 8, 2480–2493 (2022).
Medline CASGoogle Scholar
34. Tyagi P, Singh M, Kumari H, Kumari A, Mukhopadhyay K. Bactericidal activity of curcumin I is associated with damaging of bacterial membrane. PLOS ONE 10(3), e0121313 (2015).
MedlineGoogle Scholar
35. Bessa LJ, Eaton P, Dematei A et al. Synergistic and antibiofilm properties of ocellatin peptides against multidrug-resistant Pseudomonas aeruginosa. Future Microbiol. 13(2), 151–163 (2018).
CASGoogle Scholar
36. Galdiero E, Lombardi L, Falanga A, Libralato G, Guida M, Carotenuto R. Biofilms: novel strategies based on antimicrobial peptides. Pharmaceutics 11(7), 322 (2019).
Medline CASGoogle Scholar
37. Rodríguez-Rojas A, Rolff J. Antimicrobial activity of cationic antimicrobial peptides against stationary phase bacteria. Front. Microbiol. 13, 1029084 (2022).
MedlineGoogle Scholar
38. Karslake J, Maltas J, Brumm P, Wood KB. Population density modulates drug inhibition and gives rise to potential bistability of treatment outcomes for bacterial infections. PLoS Comput. Biol. 12(10), e1005098 (2016).
MedlineGoogle Scholar
39. Holmes CL, Anderson MT, Mobley HLT, Bachman MA. Pathogenesis of Gram-negative bacteremia. Clin. Microbiol. Rev. 34, e00234–20 (2021).
Medline CASGoogle Scholar
40. Masadeh MM, Laila SA, Haddad R, Alzoubi K, Alhaijaa AA, Alrabadi N. The antimicrobial effect against multi-drug resistant bacteria of the SK4 peptide: a novel hybrid peptide of cecropin-A and BMAP-27. Curr. Pharm. Biotechnol. 24(8), 1070–1078 (2023).
Medline CASGoogle Scholar
41. Selvarajan V, Tram NDT, Xu J et al. Stapled β-hairpin antimicrobial peptides with improved stability and activity against drug-resistant Gram-negative bacteria. J. Med. Chem. 66(13), 8498–8509 (2023).
Medline CASGoogle Scholar
42. Madhuri, Shireen T, Venugopal S et al. In vitro antimicrobial activity of alpha-melanocyte stimulating hormone against major human pathogen Staphylococcus aureus. Peptides 30(9), 1627–1635 (2009).
Medline CASGoogle Scholar
43. Grieco P, Carotenuto A, Auriemma L, Limatola A, Di Maro D. Novel α-MSH peptide analogues with broad spectrum antimicrobial activity. PLOS ONE 8(4), e61614 (2013).
Medline CASGoogle Scholar
44. Lyu Y, Tan M, Xue M et al. Broad-spectrum hybrid antimicrobial peptides derived from PMAP-23 with potential LPS binding ability. Biochem. Pharmacol. 210, 115500 (2023).
Medline CASGoogle Scholar
45. Schmidtchen A, Malmsten M. Peptide interactions with bacterial lipopolysaccharides. Curr. Opin. Colloid Interface Sci. 18(5), 381–392 (2013).
CASGoogle Scholar
46. Dong W, Mao X, Guan Y, Kang Y, Shang D. Antimicrobial and anti-inflammatory activities of three chensinin-1 peptides containing mutation of glycine and histidine residues. Sci. Rep. 7, 40228 (2017).
Medline CASGoogle Scholar
47. Zanin LMP, Alvares DDS, Juliano MA, Pazin WM, Ito AS, Neto JR. Interaction of a synthetic antimicrobial peptide with model membrane by fluorescence spectroscopy. Eur. Biophys. J. 42(11–12), 819–831 (2013).
Medline CASGoogle Scholar
48. Dathe M, Nikolenko H, Klose J, Bienert M. Cyclization increases the antimicrobial activity and selectivity of arginine- and tryptophan-containing hexapeptides. Biochemistry 43(28), 9140–9150 (2004).
Medline CASGoogle Scholar
49. Grishina IB, Woody RW. Contributions of tryptophan side chains to the circular dichroism of globular proteins: exciton couplets and coupled oscillators. Faraday Discuss. 99, 245–262 (1994).
CASGoogle Scholar
50. Wang J, Chou S, Xu L et al. High specific selectivity and membrane-active mechanism of the synthetic centrosymmetric α-helical peptides with Gly–Gly pairs. Sci. Rep. 5, 15963 (2015).
Medline CASGoogle Scholar
51. Li Q, Cebrián R, Montalbán-López M, Ren H, Wu W, Kuipers OP. Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens. Commun. Biol. 4, 31 (2021).
Medline CASGoogle Scholar
52. Zhu X, Shan A, Ma Z et al. Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 59, 3008–3017 (2015).
Medline CASGoogle Scholar
53. Lv Y, Wang J, Gao H, Wang Z, Dong N. Antimicrobial properties and membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin PMAP-36. PLOS ONE 9(1), e86364 (2014).
MedlineGoogle Scholar

Vol. 19, No. 3

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Received 6 April 2023

Accepted 20 October 2023

Published online 21 December 2023

Published in print February 2024

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Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/fmb-2023-0080

Author contributions

K Tiwari and P Patel contributed equally to this work. K Mukhopadhyay and K Tiwari conceived and designed the study and wrote the manuscript. K Tiwari and P Patel performed the experiments, analyzed data and prepared the figures and tables. A H Mondal conceived and designed the synergy experiments. All authors reviewed the manuscript. The present address of A H Mondal is “Department of Microbiology, Faculty of Allied Health Sciences, Shree Guru Gobind Singh Tricentenary University, Gurugram-122505 Haryana, India”.

Acknowledgments

The authors are thankful to B Dhawan, AIIMS, New Delhi for providing clinical isolates of E. coli. They thank S Mumtaz for critical reading and editing the manuscript. They thank M Singh (SES, JNU) for the use of instruments obtained from her grant DST WOS-A (SR/WOS-A/LS-36/2018), M Vashistha and R Pal (AIRF, JNU), for helping in acquisition of TEM and SEM images. They also acknowledge Department of Biotechnology for funding AIRF, JNU for instrumental facilities.

Financial disclosure

K Mukhopadhyay is thankful for receiving a financial grant from the Department of Biotechnology (BT/PR27737/MED/29/1265/2018). K Tiwari and P Patel are thankful to the funding agency UGC for their research fellowships. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

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