STRUCTURAL, FUNCTIONAL AND EVOLUTIONARY STUDIES OF ANTIMICROBIAL PEPTIDES

Antimicrobial peptides represent a heterogeneous group that displays multiple modes of action such as bacteriostatic, microbicidal and cytolytic properties that are sequence and concentration dependent. Life threatening infectious disease is now a worldwide crisis and treating them effectively is becoming difficult day by day, due to the emergence of antibiotic resistant strains at alarming rates. Hence, there is an urgent need for new class of antibiotics and, antimicrobial peptides (AMPs) are an ideal candidate for this job. AMPs are gene encoded short (<100 amino acids), amphipathic molecules with hydrophobic and cationic amino acids arranged spatially which exhibit broad-spectrum antimicrobial activity. AMPs form an ancient non-specific type of innate immunity found universally in all living organisms and used as the principal first line of defense against the invading pathogen. AMPs have been in the process of evolution, as have the microbes, for hundreds of years. Despite the long history of co-evolution, AMPs have not lost their ability to kill the microbes totally nor have the microbes learnt to avoid the lethal punch of AMPs. Based upon accumulating positive data, we are encouraged to believe that antimicrobial peptides have a great potential to be the next breakthrough and first novel, truly biological in nature, class of antibiotics. The purpose of this study was twofold; primarily to elucidate the factors involved in governing the peptide activity and toxicity against membranes, and secondly to design a simple approach where we can boost and spread the spectrum of antimicrobial activity against pathogens such as S. aureus and P. aeruginosa for a peptide that is otherwise non-lethal to the bacteria. Results presented in this thesis show that antimicrobial domains of the anaphylatoxin C3a are structurally and evolutionary conserved. Moreover antimicrobial activity is not governed by a single factor, but instead by a combination of net charge, amphipathicity and helicity. By utilizing a low number of amino acid substitutions at strategic positions in an antimicrobial peptide derived from C3a, CNY20, we were able to develop peptides, which exert a significant activity on both S. aureus and C. albicans in contrast to the parent peptide. Although, antimicrobial activity is not governed by single parameter, the activity can still be boosted by end-tagging of a peptide with hydrophobic oligopeptide stretches. This modification promotes peptide binding to bacteria and subsequent cell wall rupture, but does not increase the toxicity or the protease susceptibility of the peptide. It is noteworthy that end tagging of ultra short peptides spanning 5-7 amino acids with hydrophobic amino acids enhances bactericidal activity, while preserving low toxicity and protease resistance

Antimicrobial activity of a C-terminal peptide from human extracellular superoxide dismutase

<p>Abstract</p> <p>Background</p> <p>Antimicrobial peptides (AMP) are important effectors of the innate immune system. Although there is increasing evidence that AMPs influence bacteria in a multitude of ways, bacterial wall rupture plays the pivotal role in the bactericidal action of AMPs. Structurally, AMPs share many similarities with endogenous heparin-binding peptides with respect to secondary structure, cationicity, and amphipathicity.</p> <p>Findings</p> <p>In this study, we show that RQA21 (RQAREHSERKKRRRESECKAA), a cationic and hydrophilic heparin-binding peptide corresponding to the C-terminal region of extracellular superoxide dismutase (SOD), exerts antimicrobial activity against <it>Escherichia coli</it>, <it>Pseudomonas aeruginosa</it>, <it>Staphylococcus aureus</it>, <it>Bacillus subtilis </it>and <it>Candida albicans</it>. The peptide was also found to induce membrane leakage of negatively charged liposomes. However, its antibacterial effects were abrogated in physiological salt conditions as well as in plasma.</p> <p>Conclusion</p> <p>The results provide further evidence that heparin-binding peptide regions are multifunctional, but also illustrate that cationicity alone is not sufficient for AMP function at physiological conditions. However, our observation, apart from providing a link between heparin-binding peptides and AMPs, raises the hypothesis that proteolytically generated C-terminal SOD-derived peptides could interact with, and possibly counteract bacteria. Further studies are therefore merited to study a possible role of SOD in host defence.</p

Supplementary data for the article: Rajan, S.; Pattanaik, A.; Kumaresan, V.; Bhatt, P.; Gunasekaran, S.; Arockiaraj, J.; Pasupuleti, M.; Beškoski, V. P.; Chakraborty, P. Characterization of Some Naphthalene Using Bacteria Isolated from Contaminated Cooum Riverine Sediment of the Bay of Bengal (India). Journal of the Serbian Chemical Society 2019, 84 (2), 225–236. https://doi.org/10.2298/JSC180724088R

Supplementary material for: [https://doi.org/10.2298/JSC180724088R]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3048

Antimicrobial Activity of Human Prion Protein Is Mediated by Its N-Terminal Region

BACKGROUND: Cellular prion-related protein (PrP(c)) is a cell-surface protein that is ubiquitously expressed in the human body. The multifunctionality of PrP(c), and presence of an exposed cationic and heparin-binding N-terminus, a feature characterizing many antimicrobial peptides, made us hypothesize that PrP(c) could exert antimicrobial activity. METHODOLOGY AND PRINCIPAL FINDINGS: Intact recombinant PrP exerted antibacterial and antifungal effects at normal and low pH. Studies employing recombinant PrP and N- and C-terminally truncated variants, as well as overlapping peptide 20mers, demonstrated that the antimicrobial activity is mediated by the unstructured N-terminal part of the protein. Synthetic peptides of the N-terminus of PrP killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungus Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. In contrast to LL-37, however, no marked helix induction was detected for the PrP-derived peptides in presence of negatively charged (bacteria-mimicking) liposomes. PrP furthermore showed an inducible expression during wounding of human skin ex vivo and in vivo, as well as stimulation of keratinocytes with TGF-alpha in vitro. CONCLUSIONS: The demonstration of an antimicrobial activity of PrP, localisation of its activity to the N-terminal and heparin-binding region, combined with results showing an increased expression of PrP during wounding, indicate that PrPs could have a previously undisclosed role in host defense

Characterization of some naphthalene using bacteria isolated from contaminated Cooum Riverine sediment of the Bay of Bengal (India)

Microorganisms capable of using naphthalene as the sole carbon source were isolated from the contaminated sediment of Cooum River. Twenty one isolates were recovered and nine were selected for enrichment due to differences in their morphological characteristics. Out of nine isolates, only four (NS3-SRMND14B, NS14-SRMND14A, NS15-SRMND14D and NS19- -SRMND14E) were capable of completely utilizing naphthalene as the sole source of carbon in carbon free minimal medium (CFMM) supplemented with naphthalene. 16S rDNA sequencing showed that all the four isolates were distantly related to each other and belongs to Bacillus sp. (NS3-SRMND14B), Pseudomonas sp. (NS14-SRMND14A), Cellulosimicrobium sp. (NS15-SRMND14D) and Sphingobacterium sp. (NS19-SRMND14E), respectively. Based on the phylogenetic analysis of 16S rDNA sequencing, the isolate Sphingobacterium sp. (NS19-SRMND14E) has been identified as novel strain. Polymerase chain reaction (PCR) technique showed the presence of naphthalene dioxygenase (ndo) gene responsible for naphthalene degradation only in the Pseudomonas sp. (NS14-SRMND14A). We observed that the ndo gene is not the only gene responsible for naphthalene degradation. Based on our study, the indigenous microorganisms isolated from Cooum Riverine sediment can be used for bioremediation of the polluted sediment along the Bay of Bengal.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3840

Peroxiredoxin of Arthrospira platensis derived short molecule YT12 influences antioxidant and anticancer activity

This study demonstrates both the antioxidant and anticancer potential of the novel short molecule YT12 derived from peroxiredoxin (Prx) of spirulina, Arthrospira platensis (Ap). ApPrx showed significant reduction in reactive oxygen species (ROS) against hydrogen peroxide (H2O2) stress. The complementary DNA sequence of ApPrx contained 706 nucleotides and its coding region possessed 546 nucleotides between position 115 and 660. Real-time quantitative reverse transcription polymerase chain reaction analysis confirmed the messenger RNA expression of ApPrx due to H2O2 exposure in spirulina cells at regular intervals, in which the highest expression was noticed on Day 20. Cytotoxicity assay was performed using human peripheral blood mononuclear cells, and revealed that at 10 μM, the YT12 did not exhibit any notable toxicity. Furthermore, ROS scavenging activity of YT12 was performed using DCF-DA assay, in which YT12 scavenged a significant amount of ROS at 25 μM in H2O2-treated blood leukocytes. The intracellular ROS in human colon adenocarcinoma cells (HT-29) was regulated by oxidative stress, where the YT12 scavenges ROS in HT-29 cells at 12.5 μM. Findings show that YT12 peptide has anticancer activity, when treated against HT-29 cells. Through the MTT assay, YT12 showed vital cytotoxicity against HT-29 cells. These finding suggested that YT12 is a potent antioxidant molecule which defends ROS against oxidative stress and plays a role in redox balance

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

Background: Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. Methodology and Principal Findings: Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various "superbugs'' including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. Conclusions/Significance: Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise "tuning'' of toxicity and proteolytic stability may be achieved by changing tag-length and adding W-or F-amino acid tags

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion

End-Tagging of Ultra-Short Antimicrobial Peptides by W/F Stretches to Facilitate Bacterial Killing

BACKGROUND: Due to increasing resistance development among bacteria, antimicrobial peptides (AMPs), are receiving increased attention. Ideally, AMP should display high bactericidal potency, but low toxicity against (human) eukaryotic cells. Additionally, short and proteolytically stable AMPs are desired to maximize bioavailability and therapeutic versatility. METHODOLOGY AND PRINCIPAL FINDINGS: A facile approach is demonstrated for reaching high potency of ultra-short antimicrobal peptides through end-tagging with W and F stretches. Focusing on a peptide derived from kininogen, KNKGKKNGKH (KNK10) and truncations thereof, end-tagging resulted in enhanced bactericidal effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Through end-tagging, potency and salt resistance could be maintained down to 4-7 amino acids in the hydrophilic template peptide. Although tagging resulted in increased eukaryotic cell permeabilization at low ionic strength, the latter was insignificant at physiological ionic strength and in the presence of serum. Quantitatively, the most potent peptides investigated displayed bactericidal effects comparable to, or in excess of, that of the benchmark antimicrobial peptide LL-37. The higher bactericidal potency of the tagged peptides correlated to a higher degree of binding to bacteria, and resulting bacterial wall rupture. Analogously, tagging enhanced peptide-induced rupture of liposomes, particularly anionic ones. Additionally, end-tagging facilitated binding to bacterial lipopolysaccharide, both effects probably contributing to the selectivity displayed by these peptides between bacteria and eukaryotic cells. Importantly, W-tagging resulted in peptides with maintained stability against proteolytic degradation by human leukocyte elastase, as well as staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo for pig skin infected by S. aureus and E. coli. CONCLUSIONS/SIGNIFICANCE: End-tagging by hydrophobic amino acid stretches may be employed to enhance bactericidal potency also of ultra-short AMPs at maintained limited toxicity. The approach is of general applicability, and facilitates straightforward synthesis of hydrophobically modified AMPs without the need for post-peptide synthesis modifications

Effect of hydrophobic modifications in antimicrobial peptides.

With increasing resistance development against conventional antibiotics, there is an urgent need to identify novel approaches for infection treatment. Antimicrobial peptides may offer opportunities in this context, hence there has been considerable interest in identification and optimization of such peptides during the last decade in particular, with the long-term aim of developing these to potent and safe therapeutics. In the present overview, focus is placed on hydrophobic modifications of antimicrobial peptides, and how these may provide opportunities to combat also more demanding pathogens, including multi-resistant strains, yet not provoking unacceptable toxic responses. In doing so, physicochemical factors affecting peptide interactions with bacterial and eukaryotic cell membranes are discussed. Throughout, an attempt is made to illustrate how physicochemical studies on model lipid membranes can be correlated to result from bacterial and cell assays, and knowledge from this translated into therapeutic considerations