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Antimicrobial peptides are an evolutionarily conserved component of the innate immune response and are found among all classes of life.
- Describe the role of antimicrobial peptides in host defense
- Antimicrobial peptides are a unique and diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure.
- The modes of action by which antimicrobial peptides kill bacteria is varied and includes disrupting membranes, interfering with metabolism, and targeting cytoplasmic components.
- Antimicrobial peptides have been demonstrated to have a number of immunomodulatory functions that may be involved in the clearance of infection.
- antimicrobial peptide: Antimicrobial peptides (also called host defense peptides) are an evolutionarily conserved component of the innate immune response and are found among all classes of life.
- innate immune: The innate immune system, also known as non-specific immune system and first line of defense, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
- molecules: A molecule is an electrically neutral group of two or more atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their lack of electrical charge. However, in quantum physics, organic chemistry, and biochemistry, the term molecule is often used less strictly, also being applied to polyatomic ions.
Antimicrobial peptides (also called host defense peptides) are an evolutionarily conserved component of the innate immune response and are found among all classes of life. Fundamental differences exist between prokaryotic and eukaryotic cells that may represent targets for antimicrobial peptides. These peptides are potent, broad spectrum antibiotics which demonstrate potential as novel therapeutic agents. Antimicrobial peptides have been demonstrated to kill Gram negative and Gram positive bacteria (including strains that are resistant to conventional antibiotics), mycobacteria (including Mycobacterium tuberculosis), enveloped viruses, fungi and even transformed or cancerous cells. Unlike the majority of conventional antibiotics, it appears as though antimicrobial peptides may also have the ability to enhance immunity by functioning as immunomodulators.
Antimicrobial peptides are a unique and diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure. Antimicrobial peptides generally consist of between 12 and 50 amino acids. These peptides include two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion (generally >50%) of hydrophobic residues. The secondary structures of these molecules follow 4 themes, including:
Various AMPs: These are various antimicrobial peptide structures.
- β-stranded due to the presence of 2 or more disulfide bonds
- β-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain
Many of these peptides are unstructured in free solution, and fold into their final configuration upon partitioning into biological membranes. It contains hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. This amphipathicity of the antimicrobial peptides allows the partition of the membrane lipid bilayer. The ability to associate with membranes is a definitive feature of antimicrobial peptides, although membrane permeabilisation is not necessary. These peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets.
The modes of action by which antimicrobial peptides kill bacteria is varied and includes disrupting membranes, interfering with metabolism, and targeting cytoplasmic components. The initial contact between the peptide and the target organism is electrostatic, as most bacterial surfaces are anionic, or hydrophobic, such as in the antimicrobial peptide Piscidin. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’ or ‘toroidal-pore’ mechanisms. Alternately, they may penetrate into the cell to bind intracellular molecules which are crucial to cell living. Intracellular binding models include inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of DNA, RNA, and protein synthesis, and inhibition of certain enzymes. However, in many cases, the exact mechanism of killing is not known. One emerging technique for the study of such mechanisms is dual polarisation interferometry. In contrast to many conventional antibiotics these peptides appear to be bacteriocidal (bacteria killing) instead of bacteriostatic (bacteria growth inhibiting). In general the antimicrobial activity of these peptides is determined by measuring the minimal inhibitory concentration (MIC), which is the lowest concentration of drug that inhibits bacterial growth.
In addition to killing bacteria directly, they have been demonstrated to have a number of immunomodulatory functions that may be involved in the clearance of infection, including the ability to:
- Alter host gene expression
- Act as chemokines and/or induce chemokine production,
- Inhibit lipopolysaccharide induced pro-inflammatory cytokine production
- Promote wound healing
- Modulate the responses of dendritic cells and cells of the adaptive immune response
Animal models indicate that host defense peptides are crucial for both prevention and clearance of infection. It appears as though many peptides initially isolated and termed as “antimicrobial peptides” have been shown to have more significant alternative functions in vivo (e.g. hepcidin).
Several methods have been used to determine the mechanisms of antimicrobial peptide activity. In particular, solid-state NMR studies have provided an atomic-level resolution explanation of membrane disruption by antimicrobial peptides.
Antimicrobial peptides of multicellular organisms
Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?
Epinecidin-1 Has Immunomodulatory Effects, Facilitating Its Therapeutic Use in a Mouse Model of Pseudomonas aeruginosa SepsisFIG 1 Effects of epinecidin-1 (epi) treatment on mice infected with P. aeruginosa R or P. aeruginosa ATCC 19660. (A and B) Mice were injected with P. aeruginosa ATCC 19660 (A) or P. aeruginosa R (B), and independent groups (n = 27) were subsequently injected with epinecidin-1, clarithromycin, or imipenem. The survival rate was monitored on a daily basis for up to 7 days. (C and D) To determine the curative potential, mice were first injected with P. aeruginosa ATCC 19660 (C) or P. aeruginosa R (D) and then with epinecidin-1 (0.005 mg/g) 10, 60, 120, 180, or 360 min later. At these injection times, the P. aeruginosa ATCC 19660 experimental groups exhibited survival rates of 93.3%, 73.3%, 60.0%, 46.6%, and 33.3%, respectively, while the P. aeruginosa R experimental groups exhibited survival rates of 93.3%, 60.0%, 60.0%, 46.6%, and 20.0%, respectively.
|Strain and treatment||% lethality||Mean ± SD bacterial count (CFU/ml) in:|
|Blood||Peritoneum||Spleen||Liver||Mesenteric lymph nodes|
|P. aeruginosa ATCC 19660|
|No treatment||93.34 C||8.9 × 10 6 ± 2.4 × 10 6B||2.6 × 10 9 ± 1.0 × 10 9B||2.8 × 10 9 ± 1.6 × 10 9B||3.3 × 10 9 ± 1.0 × 10 9B||4.3 × 10 9 ± 2.1 × 10 9B|
|Clarithromycin (0.01 mg/g)||95.34 D||8.9 × 10 6 ± 1.0 × 10 6B||3.4 × 10 9 ± 1.4 × 10 9B||2.6 × 10 9 ± 1.2 × 10 9B||3.6 × 10 9 ± 2.0 × 10 9B||4.3 × 10 9 ± 1.0 × 10 9B|
|Imipenem (0.01 mg/g)||0 A||0 A||1.7 × 10 6 ± 1.0 × 10 6A||2.2 × 10 6 ± 0.6 × 10 6A||2.8 × 10 6 ± 1.0 × 10 6A||1.8 × 10 6 ± 1.0 × 10 6A|
|Epinecidin-1 (5 μg/g)||5 B||0 A||3.1 × 10 6 ± 1.2 × 10 6A||1.3 × 10 6 ± 1.0 × 10 6A||3.8 × 10 6 ± 1.4 × 10 6A||1.3 × 10 6 ± 1.6 × 10 6A|
|P. aeruginosa R|
|No treatment||88.6 C||7.9 × 10 6 ± 1.4 × 10 6B||4.6 × 10 9 ± 1.6 × 10 9C||2.8 × 10 9 ± 1.6 × 10 9C||3.3 × 10 9 ± 1.0 × 10 9B||4.3 × 10 9 ± 2.1 × 10 9B|
|Clarithromycin (0.01 mg/g)||88.6 C||8.4 × 10 6 ± 1.0 × 10 6B||2.4 × 10 9 ± 1.4 × 10 9C||2.6 × 10 9 ± 1.2 × 10 9C||3.6 × 10 9 ± 2.0 × 10 9B||4.3 × 10 9 ± 1.0 × 10 9B|
|Imipenem (0.01 mg/g)||60.97 B||1.7 × 10 6 ± 1.0 × 10 6B||4.7 × 10 7 ± 1.0 × 10 7B||1.2 × 10 7 ± 0.2 × 10 7B||3.6 × 10 9 ± 2.0 × 10 9B||1.7 × 10 6 ± 1.0 × 10 6A|
|Epinecidin-1 (5 μg/g)||0 A||0 A||4.1 × 10 5 ± 1.0 × 10 5A||5.3 × 10 6 ± 1.8 × 10 6A||3.5 × 10 7 ± 1.4 × 10 7A||2.4 ×0 6 ± 1.0 × 10 6A|
Toxicity and pharmacokinetics.
|Dose (mg/kg)||No. of mice, effect after receiving epinecidin-1 a|
|5||3, no effect|
|25||3, no effect|
|50||3, no effect|
|75||2, no effect 1, toxicity level 1|
|100||2, no effect 1, toxicity level 1|
Anti-inflammatory effect of epinecidin-1 on endotoxin and cytokine levels.FIG 3 Plasma levels of endotoxins and PAS ( P. aeruginosa ) in mice infected with P. aeruginosa R or ATCC 19660 after treatment with antibiotics or epinecidin-1. epi, epinecidin-1 CLE, clarithromycin IMP, imipenem. Data with different letters differ significantly (P < 0.05) between treatments. FIG 4 Plasma levels of IL-1β, IL-6, and TNF-α. Sera for ELISA were removed from the tail vein at 0, 2, 6, 12, 24, and 48 h during the treatment period. Data with different letters differ significantly (P < 0.05) between treatments.
Gene expression profiles are altered by injection of epinecidin-1.
|GO no.||Function||Count (no. of genes)||P value|
|GO:0007166||Cell surface receptor-linked signal transduction||200||0.03687171|
|GO:0007186||G-protein-coupled receptor protein signaling pathway||159||0.01237965|
|GO:0016071||mRNA metabolic process||30||0.06115789|
|GO:0034621||Cellular macromolecular complex subunit organization||28||0.01568345|
|GO:0034622||Cellular macromolecular complex assembly||27||0.00627889|
|GO:0006461||Protein complex assembly||26||0.01967148|
|GO:0070271||Protein complex biogenesis||26||0.01967148|
|GO:0032940||Secretion by cell||24||0.00674446|
|GO:0010817||Regulation of hormone levels||16||0.03861235|
|GO:0016042||Lipid catabolic process||15||0.0956397|
|GO:0003001||Generation of a signal involved in cell-cell signaling||13||0.01552742|
|GO:0033365||Protein localization in organelle||12||0.08372683|
|GO:0046942||Carboxylic acid transport||12||0.09359128|
|GO:0015931||Nucleobase, nucleoside, nucleotide, and nucleic acid transport||11||0.03636812|
|GO:0034504||Protein localization in nucleus||10||0.0278588|
|GO:0032269||Negative regulation of cellular protein metabolic process||10||0.06888556|
|GO:0006022||Aminoglycan metabolic process||8||0.0742951|
|GO:0030072||Peptide hormone secretion||6||0.09087306|
|GO:0006970||Response to osmotic stress||5||0.02885352|
|GO:0010741||Negative regulation of protein kinase cascade||5||0.09817185|
|GO:0030433||Endoplasmic reticulum-associated protein catabolic process||4||0.06021506|
Antimicrobial Peptides from Amphibian Innate Immune System as Potent Antidiabetic Agents: A Literature Review and Bioinformatics Analysis
Antimicrobial peptides, as an important member of the innate immune system, have various biological activities in addition to antimicrobial activity. There are some AMPs with antidiabetic activity, especially those isolated from amphibians. These peptides can induce insulin release via different mechanisms based on peptide type. In this review study, we collected all reported AMPs with antidiabetic activity. We also analyze the sequence and structure of these peptides for evaluation of sequence and structure effect on their antidiabetic activity. Based on this review, the biggest peptide family with antidiabetic activity is temporins with nine antidiabetic peptides. Frogs are the most abundant source of antidiabetic peptides. Bioinformatics analysis showed that an increase of positive net charge and a decrease of hydrophobicity can improve the insulinotropic effect of peptides. Peptides with higher positive net charge and Boman index showed higher activity. Based on this review article, AMPs with antidiabetic activity, especially those isolated from amphibians, can be used as novel antidiabetic drug for type 2 diabetes disease. So, amphibians are potential sources for active peptides which merit further evaluation as novel insulin secretagogues. However, strategy for the increase of stability and positive activity as well as the decrease of negative side effects must be considered.
Diabetes, as a metabolic disorder, is characterized by high blood sugar level. Types 1 and 2 are the two main types of this disorder. Between these two types, type 2 diabetes is more common than type 1 [1, 2]. Type 2 diabetes, as a chronic disease, is characterized by high levels of blood sugar. The prevalence of this disease is increasing in the world due to lifestyle changes [2, 3]. Development of effective strategies for treatment and management of type 2 diabetes is necessary . Glucose-lowering agents with natural origin can be considered for this treatment and management [5, 6]. Antimicrobial peptides are shown as important members of animal innate immune systems with broad antimicrobial activities. In addition to antimicrobial activity, these interesting peptides have various biological activities such as antiendotoxin, antiparasitic, anticancer, wound healing, spermicidal, insecticidal, and antioxidant [7–11]. The skins of amphibians produce peptides with inhibitory effects on bacterial and fungal growth . These peptides were secreted in response to stress and infection. These peptides act as the first defense system against pathogens . One of the interesting activities of some of these peptides is antidiabetic activity . Antidiabetic peptides were first reported and identified from skin secretions of amphibians. These peptides have the ability to stimulate insulin release in vitro from BRIN-BD11 rat clonal β cells at low concentrations with low cell toxicity. Approximately, more than of 99% of antidiabetic peptides have been isolated from skin secretion of amphibians, especially from the species of Anura. In this review article, we assessed all reported antidiabetic peptides in terms of source, sequence, structure, and mechanism. We also evaluated these peptides for prediction and design of new drugs for the improvement of diabetic symptoms.
All qualitative and quantitative original articles with English language about antidiabetic peptides were entered. Important databases including Iran Medex, MEDLINE/PubMed, Google Scholar, and CINAHL and other pertinent references on websites were reviewed for article selection. The used search profiles were as follows: peptide/anti-diabetic/amphibain, AMPs/anti-diabetic/amphibain, peptide/antidiabetic/amphibain, AMPs/antidiabetic/amphibain, peptide/diabet/amphibain, AMPs/diabet/amphibain, peptide/anti-diabetic/frog, AMPs/anti-diabetic/frog, peptide/antidiabetic/frog, AMPs/antidiabetic/frog, peptide/diabet/frog, AMPs/diabet/frog, peptide/anti-diabetic/toad, AMPs/anti-diabetic/toad, peptide/antidiabetic/toad, AMPs/antidiabetic/toad, peptide/diabet/toad, AMPs/diabet/toad.
Articles not directly relevant to the subject were excluded. The EndNote software was used to handle the proper references.
For bioinformatics analysis (amino acid composition analysis, alignment, structure prediction, etc.) of acquired peptides, the CLC software was used.
3. Results and Discussion
Forty-seven AMPs with antidiabetic activity were acquired by database search. Among these peptides, only two peptides were identified from insects, social wasp, Agelaia pallipes pallipes. Other peptides were isolated from different amphibians. The properties of these 45 peptides are summarized in Table 1.
3.1. Brevinin-1CBb, Brevinin-1Pa, Brevinin-1E, Brevinin-2GUb, and Brevinin-2EC
These brevinin peptides showed a significant effect on the enhancement of insulin release at a concentration of 100 nM on rat BRIN-BD11 clonal beta cell line. These peptides had no effect on intracellular calcium. These peptides have weak hemolytic activity on human erythrocytes. The proposed mechanism for the antidiabetic effect of these peptides is possible involvement of both cyclic AMP-protein kinase A- and C-dependent G-protein sensitive pathways. The insulinotropic effect of these peptides has also been attributed to their effect on Rho G proteins [14–17].
3.2. Esculentin-1, Esculentin-1b, and Esculentin-2Cha
Similar to brevinin peptides, most of esculentin peptides showed significant insulin-releasing activity. Cyclic AMP-protein kinase A- and C-dependent G-protein sensitive pathways have been proposed for the antidiabetic action of esculentin peptides [16, 18].
3.3. Temporin-DRa, Temporin-DRb, Temporin-Oe, Temporin-CBa, Temporin-Va, Temporin-Vb, Temporin-Vc, Temporin-CBf, and Temporin-TGb
Various temporins, with different amphibian sources, showed significant stimulatory effects on insulin release from clonal rat BRIN-BD11 cells. These peptides had no effect on the release of lactate dehydrogenase. Temporin-Oe and Temporin-TGb also showed significant toxicity at optimal concentration for stimulation of insulin release. These peptides had no effect on intracellular calcium. The proposed mechanism for these peptides is KATP channel-independent pathway [17, 19].
3.4. Ranatuerin-1CBa, Ranatuerin-2CBc, and Ranatuerin-2CBd
These peptides were isolated from bullfrog Lithobates catesbeianus. These peptides can stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line. Among these peptides, Ranatuerin-2CBd showed the highest stimulation of insulin release. On the other hand, among these peptides, Ranatuerin-2CBd is more cytotoxic than other peptides at optimal concentration. Based on studies, these three peptides have no effect on lactate dehydrogenase. This status indicated that these peptides preserve the integrity of the plasma membrane .
3.5. Dermaseptin B4 and Dermaseptin-LI1
Dermaseptins are considered as multifunctional AMPs. In between, Dermaseptin B4 and Dermaseptin-LI1 showed antidiabetic activity. Dermaseptin B4 and Dermaseptin-LI1 were isolated from Phyllomedusa trinitatis and Agalychnis litodryas, respectively. In glucose-responsive BRIN-BD11 cells, both peptides led to the stimulation of insulin release. The possible mechanism for induction of insulin secretion by these peptides has not been determined [20, 21].
3.6. Bombesin and Bombesin-Related Peptides
Bombesin is an AMP that is isolated from toad, Bombina variegata. Three bombesin and bombesin-related peptides with antidiabetic activity were isolated from Bombina variegate. The proposed mechanism of antidiabetic activity of bombesin and bombesin-related peptides is involvement of a cAMP-dependent, G protein-insensitive pathway .
3.7. Xenopsin and Xenopsin-AM2
Xenopsin and xenopsin-AM2 (containing the substitution Lys (3) ⟶ Arg in xenopsin) were isolated from X. laevis and X. amieti secretions, respectively. These two peptides lead to significant stimulations of insulin release from the rat BRIN-BD11 clonal β cell line. These peptides have no effect on the release of lactate dehydrogenase .
3.8. Palustrin-2CBa and Palustrin-1c
Palustrin-2CBa and Palustrin-1c were identified from skin secretions of Lithobates catesbeianus and Lithobates palustris, respectively. These peptides lead to significant stimulation of insulin release from BRIN-BD11 cells at low concentration [17, 24].
A member of the phylloseptin family, Phylloseptin-L2, has potent activity for the increase of insulin release from the rat BRIN-BD11 clonal beta cell line. This peptide did not stimulate the release of the cytosolic enzyme, lactate dehydrogenase. This activity was maintained in the absence of extracellular Ca(2+). The proposed mechanism for this peptide for insulin release is independent of primary involvement influx of Ca(2+) or closure of ATP-sensitive K(+) channels .
RK-13 is a 13-amino-acid insulinotropic peptide isolated from skin secretions of Agalychnis calcarifer. This peptide stimulated insulin release in a dose-dependent, glucose-sensitive manner, exerting its effects through a cyclic AMP-protein kinase A pathway independent of pertussis toxin-sensitive G proteins .
Pseudin-2 is a cationic peptide that is isolated from the skin of Pseudis paradoxa. This peptide leads to insulin release from the BRIN-BD11 clonal beta cell line without an increase of lactate dehydrogenase release. There is a mechanism involving Ca 2+ -independent pathways for this action of peptides .
GM-14 is an insulin tropic peptide that originated from Bombina variegate. This peptide increases the insulin release via involvement of a cAMP-dependent, G protein-insensitive pathway .
IN-21 was known as an insulinotropic peptide isolated from skin secretions of Bombina variegate. This peptide increases the insulin release via involvement of a cAMP-dependent, G protein-insensitive pathway .
Ocellatin-L2 is a glycine-leucine-rich peptide. This peptide was identified from norepinephrine-stimulated skin secretions of Leptodactylus laticeps. In higher concentration than 1 mM, this peptide can induce significant increase of the rate of insulin release from rat clonal BRIN-BD11 beta cells without significant effects on lactate dehydrogenase .
Plasticin-L1 is a glycine-leucine-rich peptide. This peptide has a potent stimulatory effect on insulin release from rat clonal BRIN-BD11 beta cells. Plasticin-L1 has no effect on lactate dehydrogenase .
Tigerinin-1R is a cyclic dodecapeptide isolated from skin secretion of Hoplobatrachus rugulosus. This peptide has low cytotoxicity and hemolytic activity. Tigerinin-1R could significantly stimulate the insulin release from BRIN-BD11 cells. A study in a mouse model showed that this peptide increases insulin release and improves glucose tolerance .
A caerulein-related peptide, Caerulein-B1, was identified from skin secretion of Xenopus borealis. This peptide has an additional Gly amino acid in comparison with caerulein and caerulein-B2. This peptide showed potent effect on stimulations of insulin release from the rat BRIN-BD11 clonal β cell line at low concentration .
Amolopin is an AMP with the highest similarity to temporins and vespid chemotactic peptides. This peptide showed a stimulatory effect on insulin release in INS-1 cells. Evaluation of mechanism indicated that the stimulatory effect had no effect on the increase of the influx of Ca 2+ .
Alyteserin-2a, a peptide isolated from Alytes obstetricans, has high antidiabetic activity and low toxicity on human blood cells. Membrane depolarization and increased intracellular Ca 2+ concentration have been considered as involved mechanisms in stimulation of insulin release .
3.20. Magainin-AM1 and Magainin-AM2
Magainin-AM1 and Magainin-AM2 are two AMPS isolated from Xenopus amieti. These peptides have antidiabetic activity. The proposed mechanism for action of these peptides is involvement of cell membrane depolarization and increase in intracellular calcium concentration. Studies also showed that Magainin-AM1 and Magainin-AM2 produced a significant improvement in glucose tolerance, insulin sensitivity, and improved beta cell functions in HFD-fed mice [32, 33].
Hymenochirin-1B has an antidiabetic effect in BRIN-BD 11 cells. This peptide leads to the stimulation of insulin release from the pancreatic beta cells by the KATP channel-independent pathway. This peptide can increase the plasma insulin level after intraperitoneal administration in HFD-fed mice .
Based on literature review, all proposed mechanisms of action for antidiabetic peptides have been summarized in Figure 1.
3.22. Pseudhymenochirin-2Pa and Pseudhymenochirin-1Pb
Pseudhymenochirin-1Pb and Pseudhymenochirin-2Pa are AMPs with stimulatory action on insulin release in BRIN-BD11 clonal β cells at low concentrations. The KATP channel-independent pathway is considered as the proposed mechanism for these peptides .
3.23. Statistical, Structural, and Bioinformatics Analysis
The most abundant source of these peptides was the various species of frogs. The range of net charge for these 45 peptides is +2-6. Except for one peptide with 39% hydrophobicity, the other reported peptides have more than 48% hydrophobicity. The size range of these peptides is 8 to 46 amino acids. Among the Rana species, the most antidiabetic peptides were extracted from Asian frog Hylarana guentheri. Among the Hylidae species, four frog species have potent antidiabetic peptides: Phyllomedusa trinitatis, Agalychnis calcarifer, Agalychnis litodryas, and Hylomantis lemur. Among the Bombinatoridae species, six species of toads (genus Bombina) have potent peptides with insulin-releasing properties. In other species, the distribution of antidiabetic peptides does not follow a specific rule. Short half-life, stimulation of immune system, hemolysis, and toxicity are limitations for peptide use as antidiabetic agents. For example, Ocellatin-L2 has high hemolytic activity in addition to high activity in stimulating insulin release . The change of amino acid content can lead to improved peptide ability. Related study showed that increase of positive net charge can increase the insulin releasing potency . However, this change of net charge does not always lead to the same result. For example, increasing the net charge of peptide Pseudin-2 had no significant stimulatory effect on insulin release . On the other hand, hydrophobicity is the most important property of antimicrobial peptides studies showed that the reduction of hydrophobicity had no effect on insulinotropic effect of peptide, but decrease the hemolytic activity. So the decrease of hydrophobicity can be considered as a potent strategy for reduction of hemolytic activity without change of the antidiabetic activity of peptides . Among all reported antidiabetic peptides, Magainin-AM2, with the lowest hydrophobicity (39%), showed the lowest hemolytic activity. In some peptides, α-amidation in the C-terminal region leads to the enhancement of antidiabetic potency. This α-amidation improves possible mechanism of action including peptide-involved membrane depolarization and an increase in intracellular Ca 2+ concentration . Owolabi et al. indicated that an increase of positive net charge of AMPs by adding cationic amino acids improved the potency of the insulinotropic activity . This group also showed that an increase of hydrophobicity leads to a decrease of potency of the insulinotropic activity. The average length of all 45 peptides is 27.81 residues. The average net charge of all 45 peptides is 3.50. In Figure 2, the sequence alignment of the reported antidiabetic peptides is shown. Based on this figure, Ala, Gly, Lys, and Leu are consensus amino acids in all peptides. Bioinformatics analysis also showed that Leu, Gly, and Ala have the highest frequency in all reported antidiabetic peptides. The frequency of aromatic amino acids (Trp, Tyr, and Phe), His, Met, and Cys is low in these 45 peptides (Table 2). The Boman index is defined as the sum of the solubility values for all amino acids in sequence of peptide. This index predicts the potential of a peptide to bind to other proteins. The Boman index that is higher than 2.48 indicated high binding potential. Among the reported antidiabetic AMPs, peptides with higher Boman index have higher antidiabetic activity. This relationship is unknown. But it may be due to higher peptide ability to binding involved enzymes in insulin secretion and glucose uptake. Another strategy for the enhancement of half-life of antidiabetic peptides is substitution of L-amino acid with D-amino acid. Due to the small sizes and rapid elimination via the kidney, this strategy does not do well to solve the limitation of the peptide use . Protection of peptide with various polymer coatings can be used for the enhancement of the therapeutic index of antidiabetic peptides . This method is considered as a successful strategy for the improvement of peptide action, especially in the increase of their stability and reduction of their immunogenicity . Increase of stability is related to the increase of peptide sizes that reduce rapid renal clearance. Conjugation with polyethylene glycol (PEGylation) [41, 42], anionic polypeptide-XTEN (864 amino acid-peptides containing A, E, G, P, S, and T) [43, 44], and hyaluronic acid (HAylation) [45, 46] are mostly used for physical shielding of antidiabetic peptides. Covalent and noncovalent interaction of peptide with serum albumins is also considered for modification of antidiabetic peptides [47–49].
3.24. Animal Studies
There are some studies about antidiabetic evaluation of the mentioned AMPs in animal models. Evaluation of these studies shows that all articles used low concentrations of peptides (nmol/kg body weight) in animal models [50, 51]. The literature review showed that the significant toxicity of AMPs (IC50) can occur in higher concentrations (micromole/kg body weight) [10, 52, 53]. So these tested peptides had antidiabetic activity at concentrations that are not toxic to the cells and animal model. However, based on in vivo studies, the abovementioned AMPs had low in vivo activity due to short blood circulation time. Increase of plasma stability and blood circulation time can improve the in vivo activity of AMPs. Physical shielding and attachment of albumin or antibody are the two main proposed strategies for the increase of plasma stability and blood circulation time of AMPs .
Based on this review study, antimicrobial peptides that originated from skin secretions of frogs and toads can stimulate insulin release. These natural peptides may be useful for type 2 diabetes treatment as complementary drugs. For improvement of antidiabetic activity, increase of half-life, reduction of toxicity on blood cell and other normal cells, change of net charge and hydrophobicity via change of amino acid composition, physical shielding of peptide with various polymers, and covalent or noncovalent conjugation with albumin can be considered as a model for preparation of synthetic peptides with antidiabetic activity. On the other hand, these natural peptides can be considered as a model for preparation of synthetic peptides with antidiabetic activity.
Conflicts of Interest
None of the authors have any conflicts of interest.
Hossein Soltaninejad and Hadi Zare-Zardini are co-first authors.
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Copyright © 2021 Hossein Soltaninejad et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
RiPPs consist of any peptides (i.e. molecular weight below 10 kDa) that are ribosomally-produced and undergo some degree of enzymatic post-translational modification. This combination of peptide translation and modification is referred to as "post-ribosomal peptide synthesis" (PRPS) in analogy with nonribosomal peptide synthesis (NRPS).
Historically, the current sub-classes of RiPPs were studied individually, and common practices in nomenclature varied accordingly in the literature. More recently, with the advent of broad genome sequencing, it has been realized that these natural products share a common biosynthetic origin. In 2013, a set of uniform nomenclature guidelines were agreed upon and published by a large group of researchers in the field.  Prior to this report, RiPPs were referred to by a variety of designations, including post-ribosomal peptides, ribosomal natural products, and ribosomal peptides.
The acronym "RiPP" stands for "ribosomally synthesized and post-translationally modified peptide".
RiPPs constitute one of the major superfamilies of natural products, like alkaloids, terpenoids, and nonribosomal peptides, although they tend to be large, with molecular weights commonly in excess of 1000 Da.  The advent of next-generation sequencing methods has made genome mining of RiPPs a common strategy.  In part due to their increased discovery and hypothesized ease of engineering, the use of RiPPs as drugs is increasing. Although they are ribosomal peptides in origin, RiPPs are typically categorized as small molecules rather than biologics due to their chemical properties, such as moderate molecular weight and relatively high hydrophobicity.
The uses and biological activities of RiPPs are diverse.
RiPPs in commercial use include nisin, a food preservative, thiostrepton, a veterinary topical antibiotic, and nosiheptide and duramycin, which are animal feed additives. Phalloidin functionalized with a fluorophore is used in microscopy as a stain due to its high affinity for actin. Anantin is a RiPP used in cell biology as an atrial natriuretic peptide receptor inhibitor. 
A derivatized RiPP in clinical trials is LFF571. LFF571, a derivative of the thiopeptide GE2270-A, completed phase II clinical trials for the treatment of Clostridium difficile infections, with comparable safety and efficacy to vancomycin.   Also recently in clinical trials was the NVB302 (a derivative of the lantibiotic actagardine) which is used for the treatment of Clostridium difficile infection.  Duramycin has completed phase II clinical trials for the treatment of cystic fibrosis. 
Other bioactive RiPPs include the antibiotics cyclothiazomycin and bottromycin, the ultra-narrow spectrum antibiotic plantazolicin, and the cytotoxin patellamide A. Streptolysin S, the toxic virulence factor of Streptococcus pyogenes, is also a RiPP. Additionally, human thyroid hormone itself is a RiPP due to its biosynthetic origin as thyroglobulin.
Amatoxins and phallotoxins Edit
Amatoxins and phallotoxins are 8- and 7-membered natural products, respectively, characterized by N-to-C cyclization in addition to a tryptathionine motif derived from the crosslinking of Cys and Trp.   The amatoxins and phallotoxins also differ from other RiPPs based on the presence of a C-terminal recognition sequence in addition to the N-terminal leader peptide. α-Amanitin, an amatoxin, has a number of posttranslational modifications in addition to macrocyclization and formation of the tryptathionine bridge: oxidation of the tryptathionine leads to the presence of a sulfoxide, and numerous hydroxylations decorate the natural product. As an amatoxin, α-amanitin is an inhibitor of RNA polymerase II. 
Bottromycins contain a C-terminal decarboxylated thiazole in addition to a macrocyclic amidine. 
There are currently six known bottromycin compounds, which differ in the extent of side chain methylation, an additional characteristic of the bottromycin class. The total synthesis of bottromycin A2 was required to definitively determine the structure of the first bottromycin. 
Thus far, gene clusters predicted to produce bottromycins have been identified in the genus Streptomyces. Bottromycins differ from other RiPPs in that there is no N-terminal leader peptide. Rather, the precursor peptide has a C-terminal extension of 35-37 amino acids, hypothesized to act as a recognition sequence for posttranslational machinery. 
Cyanobactins are diverse metabolites from cyanobacteria with N-to-C macrocylization of a 6–20 amino acid chain. Cyanobactins are natural products isolated from cyanobacteria, and close to 30% of all cyanobacterial strains are thought to contain cyanobacterial gene clusters.  However, while thus far all cyanobactins are credited to cyanobacteria, there exists the possibility that other organisms could produce similar natural products.
The precursor peptide of the cyanobactin family is traditionally designated the "E" gene, whereas precursor peptides are designated gene "A" in most RiPP gene clusters. "A" is a serine protease involved in cleavage of the leader peptide and subsequent macrocyclization of the peptide natural product, in combination with an additional serine protease homologue, the encoded by gene "G". Members of the cyanobactin family may bear thiazolines/oxazolines, thiazoles/oxazoles, and methylations depending on additional modification enzymes. For example, perhaps the most famous cyanobactin is patellamide A, which contains two thiazoles, a methyloxazoline, and an oxazoline in its final state, a macrocycle derived from 8 amino acids.
Lanthipeptides are one of the most well-studied families of RiPPs. The family is characterized by the presence of lanthionine (Lan) and 3-methyllanthionine (MeLan) residues in the final natural product. There are four major classes of lanthipeptides, delineated by the enzymes responsible for installation of Lan and MeLan. The dehydratase and cyclase can be two separate proteins or one multifunctional enzyme. Previously, lanthipeptides were known as "lantipeptides" before a consensus was reached in the field. 
Lantibiotics are lanthipeptides that have known antimicrobial activity. The founding member of the lanthipeptide family, nisin, is a lantibiotic that has been used to prevent the growth of food-born pathogens for over 40 years. 
Lasso peptides Edit
Lasso peptides are short peptides containing an N-terminal macrolactam macrocycle "ring" through which a linear C-terminal "tail" is threaded.   Because of this threaded-loop topology, these peptides resemble lassos, giving rise to their name. They are a member of a larger class of amino-acid-based lasso structures. Additionally, lasso peptides are formally rotaxanes.
The N-terminal "ring" can be from 7 to 9 amino acids long and is formed by an isopeptide bond between the N-terminal amine of the first amino acid of the peptide and the carboxylate side chain of an aspartate or glutamate residue. The C-terminal "tail" ranges from 7 to 15 amino acids in length. 
The first amino acid of lasso peptides is almost invariably glycine or cysteine, with mutations at this site not being tolerated by known enzymes.  Thus, bioinformatics-based approaches to lasso peptide discovery have thus used this as a constraint.  However, some lasso peptides were recently discovered that also contain serine or alanine as their first residue. 
The threading of the lasso tail is trapped either by disulfide bonds between ring and tail cysteine residues (class I lasso peptides), by steric effects due to bulky residues on the tail (class II lasso peptides), or both (class III lasso peptides).  The compact structure makes lasso peptides frequently resistant to proteases or thermal unfolding. 
Linear azol(in)e-containing peptides Edit
Linear azole(in)e-containing peptides (LAPs) contain thiazoles and oxazoles, or their reduced thiazoline and oxazoline forms. Thiazol(in)es are the result of cyclization of Cys residues in the precursor peptide, while (methyl)oxazol(in)es are formed from Thr and Ser. Azole and azoline formation also modifies the residue in the -1 position, or directly C-terminal to the Cys, Ser, or Thr. A dehydrogenase in the LAP gene cluster is required for oxidation of azolines to azoles.
Plantazolicin is a LAP with extensive cyclization. Two sets of five heterocycles endow the natural product with structural rigidity and unusually selective antibacterial activity.  Streptolysin S (SLS) is perhaps the most well-studied and most famous LAP, in part because the structure is still unknown since the discovery of SLS in 1901. Thus, while the biosynthetic gene cluster suggests SLS is a LAP, structural confirmation is lacking.
Microcins are all RiPPs produced by Enterobacteriaceae with a molecular weight <10 kDa. Many members of other RiPP families, such as microcin B17 (LAP) and microcin J25 (Lasso peptide) are also considered microcins. Instead of being classified based on posttranslational modifications or modifying enzymes, microcins are instead identified by molecular weight, native producer, and antibacterial activity. Microcins are either plasmid- or chromosome-encoded, but specifically have activity against Enerobacteriaceae. Because these organisms are also often producers of microcins, the gene cluster contains not only a precursor peptide and modification enzymes, but also a self-immunity gene to protect the producing strain, and genes encoding export of the natural product.
Microcins have bioactivity against Gram-negative bacteria but usually display narrow-spectrum activity due to hijacking of specific receptors involved in the transport of essential nutrients.
Most of the characterized thiopeptides have been isolated from Actinobacteria.  General structural features of thiopeptide macrocycles, are dehydrated amino acids and thiazole rings formed from dehydrated serine/threonine and cyclized cysteine residues, respectively
The thiopeptide macrocycle is closed with a six-membered nitrogen-bearing ring. Oxidation state and substitution pattern of the nitrogenous ring determines the series of the thiopeptide natural product.  While the mechanism of macrocyclization is not known, the nitrogenous ring can exist in thiopeptides as a piperidine, dehydropiperidine, or a fully oxidized pyridine. Additionally, some thiopeptides bear a second macrocycle, which bears a quinaldic acid or indolic acid residue derived from tryptophan. Perhaps the most well-characterized thiopeptide, thiostrepton A, contains a dehydropiperidine ring and a second, quinaldic acid-containing macrocycle. Four residues are dehydrated during posttranslational modification, and the final natural product also bears four thiazoles and one azoline.
Other RiPPs Edit
Autoinducing Peptides (AIPs) and quorum sensing peptides are used as signaling molecules in the process called quorum sensing. AIPs are characterized by the presence of a cyclic ester or thioester, unlike other regulatory peptides that are linear. In pathogens, exported AIPs bind to extracellular receptors that trigger the production of virulence factors.  In Staphylococcus aureus, AIPs are biosynthesized from a precursor peptide composed of a C-terminal leader region, the core region, and negatively charged tail region that is, along with the leader peptide, cleaved before AIP export. 
Bacterial Head-to-Tail Cyclized Peptides refers exclusively to ribosomally synthesized peptides with 35-70 residues and a peptide bond between the N- and C-termini, sometimes referred to as bacteriocins, although this term is used more broadly. The distinctive nature of this class is not only the relatively large size of the natural products but also the modifying enzymes responsible for macrocyclization. Other N-to-C cyclized RiPPs, such as the cyanobactins and orbitides, have specialized biosynthetic machinery for macrocylization of much smaller core peptides. Thus far, these bacteriocins have been identified only in Gram-positive bacteria. Enterocin AS-48 was isolated from Enterococcus and, like other bacteriocins, is relatively resistant to high temperature, pH changes, and many proteases as a result of macrocyclization.  Based on solution structures and sequence alignments, bacteriocins appear to take on similar 3D structures despite little sequence homology, contributing to stability and resistance to degradation.
Conopeptides and other toxoglossan peptides are the components of the venom of predatory marine snails, such as the cone snails or Conus.  Venom peptides from cone snails are generally smaller than those found in other animal venoms (10-30 amino acids vs. 30-90 amino acids) and have more disulfide crosslinks.  A single species may have 50-200 conopeptides encoded in its genome, recognizable by a well-conserved signal sequence. 
Cyclotides are RiPPs with a head-to-tail cyclization and three conserved disulfide bonds that form a knotted structure called a cyclic cysteine knot motif.   No other posttranslational modifications have been observed on the characterized cyclotides, which are between 28 - 37 amino acids in size. Cyclotides are plant natural products and the different cyclotides appear to be species-specific. While many activities have been reported for cyclotides, it has been hypothesized that all are united by a common mechanism of binding to and disrupting the cell membrane. 
Glycocins are RiPPS that are glycosylated antimicrobial peptides. Only two members have been fully characterized, making this a small RiPP class.   Sublancin 168 and glycocin F are both Cys-glycosylated and, in addition, have disulfide bonds between non-glycosylated Cys residues. While both members bear S-glycosyl groups, RiPPs bearing O- or N-linked carbohydrates will also be included in this family as they are discovered.
Linaridins are characterized by C-terminal aminovinyl cysteine residues. While this posttranslational modification is also seen in the lanthipeptides epidermin and mersacidin, linaridins do not have Lan or MeLan residues. In addition, the linaridin moiety is formed from modification of two Cys residues, whereas lanthipeptide aminovinyl cysteines are formed from Cys and dehydroalanine (Dha).  The first linaridin to be characterized was cypemycin. 
Microviridins are cyclic N-acetylated trideca- and tetradecapeptides with ω-ester and/or ω-amide bonds. Lactone formation through glutamate or aspartate ω-carboxy groups and the lysine ε-amino group forms macrocycles in the final natural product.
Orbitides are plant-derived N-to-C cyclized peptides with no disulfide bonds. Also referred to as Caryophyllaceae-like homomonocyclopeptides,  orbitides are 5-12 amino acids in length and are composed of mainly hydrophobic residues. Similar to the amatoxins and phallotoxins, the gene sequences of orbitides suggest the presence of a C-terminal recognition sequence. In the flaxseed variety Linum usitatissimum, a precursor peptide was found using Blast searching that potentially contains five core peptides separated by putative recognition sequences. 
Proteusins are named after "Proteus", a Greek shape-shifting sea god. Until now, the only known members in the family of Proteusins are called polytheonamides. They were originally presumed to be nonribosomal natural products due to the presence of many D-amino acids and other non-proteinogenic amino acids. However, a metagenomic study revealed the natural products as the most extensively modified class of RiPPs known to date.  Six enzymes are responsible for installing a total of 48 posttranslational modifications onto the polytheonamide A and B precursor peptides, including 18 epimerizations. Polytheonamides are exceptionally large, as a single molecule is able to span a cell membrane and form an ion channel.  
Sactipeptides contain intramolecular linkages between the sulfur of Cys residues and the α-carbon of another residue in the peptide. A number of nonribosomal peptides bear the same modification. In 2003, the first RiPP with a sulfur-to-α-carbon linkage was reported when the structure of subtilosin A was determined using isotopically enriched media and NMR spectroscopy.  In the case of subtilosin A, isolated from Bacillus subtilis 168, the Cα crosslinks between Cys4 and Phe31, Cys7 and Thr28, and Cys13 and Phe22 are not the only posttranslational modifications the C- and N-termini form an amide bond, resulting in a circular structure that is conformationally restricted by the Cα bonds. Sactipeptides with antimicrobial activity are commonly referred to as sactibiotics (sulfur to alpha-carbon antibiotic). 
RiPPs are characterized by a common biosynthetic strategy wherein genetically-encoded peptides undergo translation and subsequent chemical modification by biosynthetic enzymes.
Common features Edit
All RiPPs are synthesized first at the ribosome as a precursor peptide. This peptide consists of a core peptide segment which is typically preceded (and occasionally followed) by a leader peptide segment and is typically
20-110 residues long. The leader peptide is usually important for enabling enzymatic processing of the precursor peptide via aiding in recognition of the core peptide by biosynthetic enzymes and for cellular export. Some RiPPs also contain a recognition sequence C-terminal to the core peptide these are involved in excision and cyclization. Additionally, eukaryotic RiPPs may contain a signal segment of the precursor peptide which helps direct the peptide to cellular compartments. 
During RiPP biosynthesis, the unmodified precursor peptide (containing an unmodified core peptide, UCP) is recognized and chemically modified sequentially by biosynthetic enzymes (PRPS). Examples of modifications include dehydration (i.e. lanthipeptides, thiopeptides), cyclodehydration (i.e. thiopeptides), prenylation (i.e. cyanobactins), and cyclization (i.e. lasso peptides), among others. The resulting modified precursor peptide (containing a modified core peptide, MCP) then undergoes proteolysis, wherein the non-core regions of the precursor peptide are removed. This results in the mature RiPP. 
Papers published prior to a recent community consensus  employ differing sets of nomenclature. The precursor peptide has been referred to previously as prepeptide, prepropeptide, or structural peptide. The leader peptide has been referred to as a propeptide, pro-region, or intervening region. Historical alternate terms for core peptide included propeptide, structural peptide, and toxin region (for conopeptides, specifically). 
Family-specific features Edit
Lanthipeptides are characterized by the presence lanthionine (Lan) and 3-methyllanthionine (MeLan) residues. Lan residues are formed from a thioether bridge between Cys and Ser, while MeLan residues are formed from the linkage of Cys to a Thr residue. The biosynthetic enzymes responsible for Lan and MeLan installation first dehydrate Ser and Thr to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. Subsequent thioether crosslinking occurs through a Michael-type addition by Cys onto Dha or Dhb. 
Four classes of lanthipeptide biosynthetic enzymes have been designated.  Class I lanthipeptides have dedicated lanthipeptide dehydratases, called LanB enzymes, though more specific designations are used for particular lanthipeptides (e.g. NisB is the nisin dehydratase). A separate cyclase, LanC, is responsible for the second step in Lan and MeLan biosynthesis. However, class II, III, and IV lanthipeptides have bifunctional lanthionine synthetases in their gene clusters, meaning a single enzyme carries out both dehydration and cyclization steps. Class II synthetases, designated LanM synthetases, have N-terminal dehydration domains with no sequence homology to other lanthipeptide biosynthetic enzymes the cyclase domain has homology to LanC. Class III (LanKC) and IV (LanL) enzymes have similar N-terminal lyase and central kinase domains, but diverge in C-terminal cyclization domains: the LanL cyclase domain is homologous to LanC, but the class III enzymes lack Zn-ligand binding domains. 
Linear azol(in)e-containing peptides Edit
The hallmark of linear azol(in)e-containing peptide (LAP) biosynthesis is the formation of azol(in)e heterocycles from the nucleophilic amino acids serine, threonine, or cysteine.   This is accomplished by three enzymes referred to as the B, C, and D proteins the precursor peptide is referred to as the A protein, as in other classes. 
The C protein is mainly involved in leader peptide recognition and binding and is sometimes called a scaffolding protein. The D protein is an ATP-dependent cyclodehydratase that catalyzes the cyclodehydration reaction, resulting in formation of an azoline ring. This occurs by direct activation of the amide backbone carbonyl with ATP, resulting in stoichiometric ATP consumption.  The C and D proteins are occasionally present as a single, fused protein, as is the case for trunkamide biosynthesis. The B protein is a flavin mononucleotide (FMN)-dependent dehydrogenase which oxidizes certain azoline rings into azoles.
The B protein is typically referred to as the dehydrogenase the C and D proteins together form the cyclodehydratase, although the D protein alone performs the cyclodehydration reaction. Early work on microcin B17 adopted a different nomenclature for these proteins, but a recent consensus has been adopted by the field as described above. 
Cyanobactin biosynthesis requires proteolytic cleavage of both N-terminal and C-terminal portions of the precursor peptide. The defining proteins are thus an N-terminal protease, referred to as the A protein, and a C-terminal protease, referred to as the G protein. The G protein is also responsible for macrocyclization.
For cyanobactins, the precursor peptide is referred to as the E peptide.  Minimally, the E peptide requires a leader peptide region, a core (structural) region, and both N-terminal and C-terminal protease recognition sequences. In contrast to most RiPPs, for which a single precursor peptide encodes a single natural product via a lone core peptide, cyanobactin E peptides can contain multiple core regions multiple E peptides can even be present in a single gene cluster.  
Many cyanobactins also undergo heterocyclization by a heterocyclase (referred to as the D protein), installing oxazoline or thiazoline moieties from Ser/Thr/Cys residues prior to the action of the A and G proteases.  The heterocyclase is an ATP-dependent YcaO homologue that behaves biochemically in the same manner as YcaO-domain cyclodehydratases in thiopeptide and linear azol(in)e-containing peptide (LAP) biosynthesis (described above).
A common modification is prenylation of hydroxyl groups by an F protein prenyltransferase. Oxidation of azoline heterocycles to azoles can also be accomplished by an oxidase domain located on the G protein. Unusual for ribosomal peptides, cyanobactins can include D-amino acids these can occur adjacent to azole or azoline residues.  The functions of some proteins found commonly in cyanobactin biosynthetic gene clusters, the B and C proteins, are unknown.
Thiopeptide biosynthesis involves particularly extensive modification of the core peptide scaffold. Indeed, due to the highly complex structures of thiopeptides, it was commonly thought that these natural products were nonribosomal peptides. Recognition of the ribosomal origin of these molecules came in 2009 with the independent discovery of the gene clusters for several thiopeptides.     
The standard nomenclature for thiopeptide biosynthetic proteins follows that of the thiomuracin gene cluster.   In addition to the precursor peptide, referred to as the A peptide, thiopeptide biosynthesis requires at least six genes. These include lanthipeptide-like dehydratases, designated the B and C proteins, which install dehydroalanine and dehydrobutyrine moieties by dehydrating Ser/Thr precursor residues. Azole and azoline synthesis is effected by the E protein, the dehydrogenase, and the G protein, the cyclodehydratase. The nitrogen-containing heterocycle is installed by the D protein cyclase via a putative [4+2] cycloaddition of dehydroalanine moieties to form the characteristic macrocycle.  The F protein is responsible for binding of the leader peptide. 
Thiopeptide biosynthesis is biochemically similar to that of cyanobactins, lanthipeptides, and linear azol(in)e-containing peptides (LAPs). As with cyanobactins and LAPs, azole and azoline synthesis occurs via the action of an ATP-dependent YcaO-domain cyclodehydratase. In contrast to LAPs, where cyclodehydration occurs via the action of two distinct proteins responsible for leader peptide binding and cyclodehydrative catalysis, these are fused into a single protein (G protein) in cyanobactin and thiopeptide biosynthesis.  However, in thiopeptides, an additional protein, designated the Ocin-ThiF-like protein (F protein) is necessary for leader peptide recognition and potentially recruiting other biosynthetic enzymes. 
Lasso peptides Edit
Lasso peptide biosynthesis requires at least three genes, referred to as the A, B, and C proteins.   The A gene encodes the precursor peptide, which is modified by the B and C proteins into the mature natural product. The B protein is an adenosine triphosphate-dependent cysteine protease that cleaves the leader region from the precursor peptide. The C protein displays homology to asparagine synthetase and is thought to activate the carboxylic acid side chain of a glutamate or aspartate residue via adenylylation. The N-terminal amine formed by the B protein (protease) then reacts with this activated side chain to form the macrocycle-forming isopeptide bond. The exact steps and reaction intermediates in lasso peptide biosynthesis remain unknown due to experimental difficulties associated with the proteins.  Commonly, the B protein is referred to as the lasso protease, and the C protein is referred to as the lasso cyclase.
Some lasso peptide biosynthetic gene clusters also require an additional protein of unknown function for biosynthesis. Additionally, lasso peptide gene clusters usually include an ABC transporter (D protein) or an isopeptidase, although these are not strictly required for lasso peptide biosynthesis and are sometimes absent.  No X-ray crystal structure is yet known for any lasso peptide biosynthetic protein.
The biosynthesis of lasso peptides is particularly interesting due to the inaccessibility of the threaded-lasso topology to chemical peptide synthesis.
The normal function of Aβ is not well understood.  Though some animal studies have shown that the absence of Aβ does not lead to any obvious loss of physiological function,   several potential activities have been discovered for Aβ, including activation of kinase enzymes,   protection against oxidative stress,   regulation of cholesterol transport,   functioning as a transcription factor,   and anti-microbial activity (potentially associated with Aβ's pro-inflammatory activity).   
The glymphatic system clears metabolic waste from the mammalian brain, and in particular amyloid beta.  Indeed, a number of proteases have been implicated by both genetic and biochemical studies as being responsible for the recognition and degradation of amyloid beta these include insulin degrading enzyme.  and presequence protease  The rate of removal is significantly increased during sleep.  However, the significance of the lymphatic system in Aβ clearance in Alzheimer's disease is unknown. 
Aβ is the main component of amyloid plaques, extracellular deposits found in the brains of people with Alzheimer's disease.  Aβ can also form the deposits that line cerebral blood vessels in cerebral amyloid angiopathy. The plaques are composed of a tangle of Aβ oligomers  and regularly ordered aggregates called amyloid fibrils  , a protein fold shared by other peptides such as the prions associated with protein misfolding diseases.
Alzheimer's disease Edit
Research suggests that soluble oligomeric forms of the peptide may be causative agents in the development of Alzheimer's disease.   It is generally believed that Aβ oligomers are the most toxic.  The ion channel hypothesis postulates that oligomers of soluble, non-fibrillar Aβ form membrane ion channels allowing the unregulated calcium influx into neurons  that underlies disrupted calcium ion homeostasis and apoptosis seen in Alzheimer's disease.   Computational studies have demonstrated that also Aβ peptides embedded into the membrane as monomers with predominant helical configuration, can oligomerize  and eventually form channels whose stability and conformation are sensitively correlated to the concomitant presence and arrangement of cholesterol.  A number of genetic, cell biology, biochemical and animal studies support the concept that Aβ plays a central role in the development of Alzheimer's disease pathology.  
Brain Aβ is elevated in people with sporadic Alzheimer's disease. Aβ is the main constituent of brain parenchymal and vascular amyloid it contributes to cerebrovascular lesions and is neurotoxic.     It is unresolved how Aβ accumulates in the central nervous system and subsequently initiates the disease of cells. Some researchers have found that the Aβ oligomers induce some of the symptoms of Alzheimer's disease by competing with insulin for binding sites on the insulin receptor, thus impairing glucose metabolism in the brain.  Significant efforts have been focused on the mechanisms responsible for Aβ production, including the proteolytic enzymes gamma- and β-secretases which generate Aβ from its precursor protein, APP (amyloid precursor protein).     Aβ circulates in plasma, cerebrospinal fluid (CSF) and brain interstitial fluid (ISF) mainly as soluble Aβ40   Amyloid plaques contain both Aβ40 and Aβ42,  while vascular amyloid is predominantly the shorter Aβ40. Several sequences of Aβ were found in both lesions.    Generation of Aβ in the central nervous system may take place in the neuronal axonal membranes after APP-mediated axonal transport of β-secretase and presenilin-1. 
Increases in either total Aβ levels or the relative concentration of both Aβ40 and Aβ42 (where the former is more concentrated in cerebrovascular plaques and the latter in neuritic plaques)  have been implicated in the pathogenesis of both familial and sporadic Alzheimer's disease. Due to its more hydrophobic nature, the Aβ42 is the most amyloidogenic form of the peptide. However the central sequence KLVFFAE is known to form amyloid on its own, and probably forms the core of the fibril. [ citation needed ] One study further correlated Aβ42 levels in the brain not only with onset of Alzheimer's disease, but also reduced cerebrospinal fluid pressure, suggesting that a build-up or inability to clear Aβ42 fragments may play a role into the pathology. 
The "amyloid hypothesis", that the plaques are responsible for the pathology of Alzheimer's disease, is accepted by the majority of researchers but is not conclusively established. An alternative hypothesis is that amyloid oligomers rather than plaques are responsible for the disease.   Mice that are genetically engineered to express oligomers but not plaques (APP E693Q ) develop the disease. Furthermore, mice that are in addition engineered to convert oligomers into plaques (APP E693Q X PS1ΔE9), are no more impaired than the oligomer only mice.  Intra-cellular deposits of tau protein are also seen in the disease, and may also be implicated, as has aggregation of alpha synuclein.
While Aβ has been implicated in cancer development, prompting studies on a variety of cancers to elucidate the nature of its possible effects, results are largely inconclusive. Aβ levels have been assessed in relation to a number of cancers, including esophageal, colorectal, lung, and hepatic, in response to observed reductions in risk for developing Alzheimer's disease in survivors of these cancers. All cancers were shown to be associated positively with increased Aβ levels, particularly hepatic cancers.  This direction of association however has not yet been established. Studies focusing on human breast cancer cell lines have further demonstrated that these cancerous cells display an increased level of expression of amyloid precursor protein. 
Down syndrome Edit
Adults with Down syndrome had accumulation of amyloid in association with evidence of Alzheimer's disease, including declines in cognitive functioning, memory, fine motor movements, executive functioning, and visuospatial skills. 
Aβ is formed after sequential cleavage of the amyloid precursor protein (APP), a transmembrane glycoprotein of undetermined function. APP can be cleaved by the proteolytic enzymes α-, β- and γ-secretase Aβ protein is generated by successive action of the β and γ secretases. The γ secretase, which produces the C-terminal end of the Aβ peptide, cleaves within the transmembrane region of APP and can generate a number of isoforms of 30-51 amino acid residues in length.  The most common isoforms are Aβ40 and Aβ42 the longer form is typically produced by cleavage that occurs in the endoplasmic reticulum, while the shorter form is produced by cleavage in the trans-Golgi network. 
Autosomal-dominant mutations in APP cause hereditary early-onset Alzheimer's disease (familial AD, fAD). This form of AD accounts for no more than 10% of all cases, and the vast majority of AD is not accompanied by such mutations.  However, familial Alzheimer's disease is likely to result from altered proteolytic processing. This is evidenced by the fact that many mutations that lead to fAD occur near γ-secretase cleavage sites on APP.  One of the most common mutations causing fAD, London Mutation, occurs at codon 717 of the APP gene,   and results in a valine to isoleucine amino acid substitution. Histochemical analysis of the APP V717I mutation has revealed extensive Aβ pathology throughout neuroaxis as well as wide-spread cerebral amyloid angiopathy (CAA). 
The gene for the amyloid precursor protein is located on chromosome 21, and accordingly people with Down syndrome have a very high incidence of Alzheimer's disease. 
Amyloid beta is commonly thought to be intrinsically unstructured, meaning that in solution it does not acquire a unique tertiary fold but rather populates a set of structures. As such, it cannot be crystallized and most structural knowledge on amyloid beta comes from NMR and molecular dynamics. Early NMR-derived models of a 26-aminoacid polypeptide from amyloid beta (Aβ 10-35) show a collapsed coil structure devoid of significant secondary structure content.  However, the most recent (2012) NMR structure of (Aβ 1-40) has significant secondary and tertiary structure.  Replica exchange molecular dynamics studies suggested that amyloid beta can indeed populate multiple discrete structural states  more recent studies identified a multiplicity of discrete conformational clusters by statistical analysis.  By NMR-guided simulations, amyloid beta 1-40 and amyloid beta 1-42 also seem to feature highly different conformational states,  with the C-terminus of amyloid beta 1-42 being more structured than that of the 1-40 fragment.
Low-temperature and low-salt conditions allowed to isolate pentameric disc-shaped oligomers devoid of beta structure.  In contrast, soluble oligomers prepared in the presence of detergents seem to feature substantial beta sheet content with mixed parallel and antiparallel character, different from fibrils  computational studies suggest an antiparallel beta-turn-beta motif instead for membrane-embedded oligomers. 
The suggested mechanisms by which amyloid beta may damage and cause neuronal death include the generation of reactive oxygen species during the process of its self-aggregation. When this occurs on the membrane of neurons in vitro, it causes lipid peroxidation and the generation of a toxic aldehyde called 4-hydroxynonenal which, in turn, impairs the function of ion-motive ATPases, glucose transporters and glutamate transporters. As a result, amyloid beta promotes depolarization of the synaptic membrane, excessive calcium influx and mitochondrial impairment.  Aggregations of the amyloid-beta peptide disrupt membranes in vitro. 
Researchers in Alzheimer's disease have identified several strategies as possible interventions against amyloid: 
- inhibitors. These work to block the first cleavage of APP inside of the cell, at the endoplasmic reticulum. inhibitors (e. g. semagacestat). These work to block the second cleavage of APP in the cell membrane and would then stop the subsequent formation of Aβ and its toxic fragments.
- Selective Aβ42 lowering agents (e. g. tarenflurbil). These modulate γ-secretase to reduce Aβ42 production in favor of other (shorter) Aβ versions.
β- and γ-secretase are responsible for the generation of Aβ from the release of the intracellular domain of APP, meaning that compounds that can partially inhibit the activity of either β- and γ-secretase are highly sought after. In order to initiate partial inhibition of β- and γ-secretase, a compound is needed that can block the large active site of aspartyl proteases while still being capable of bypassing the blood-brain barrier. To date, human testing has been avoided due to concern that it might interfere with signaling via Notch proteins and other cell surface receptors. [ citation needed ]
- . This stimulates the host immune system to recognize and attack Aβ, or provide antibodies that either prevent plaque deposition or enhance clearance of plaques or Aβ oligomers. Oligomerization is a chemical process that converts individual molecules into a chain consisting of a finite number of molecules. Prevention of oligomerization of Aβ has been exemplified by active or passive Aβ immunization. In this process antibodies to Aβ are used to decrease cerebral plaque levels. This is accomplished by promoting microglial clearance and/or redistributing the peptide from the brain to systemic circulation. Antibodies that target Aβ that currently in clinical trials included aducanumab, bapineuzumab, crenezumab, gantenerumab, and solanezumab.  Amyloid beta vaccines that are currently in clinical trials include CAD106 and UB-311.  However literature reviews have raised questions as to immunotherapy's overall efficacy. One such study assessing ten anti-Ab42 antibodies showed minimal cognitive protection and results within each trial, as symptoms were too far progressed by the time of application to be useful. Further development is still required for application to those who are presymptomatic
to assess their effectiveness early into disease progression. 
- Anti-aggregation agents  such as apomorphine, or carbenoxolone. The latter has commonly been used as a treatment for peptic ulcers, but also displays neuroprotective properties, shown to improve cognitive functions such as verbal fluency and memory consolidation. By binding with high affinity to Aβ42 fragments, primarily via hydrogen bonding, carbenoxolone captures the peptides before they can aggregate together, rendering them inert, as well as destabilizes those aggregates already formed, helping to clear them.  This is a common mechanism of action of anti-aggregation agents at large. 
- Studies comparing synthetic to recombinant Aβ42 in assays measuring rate of fibrillation, fibril homogeneity, and cellular toxicity showed that recombinant Aβ42 had a faster fibrillation rate and greater toxicity than synthetic amyloid beta 1-42 peptide. 
- Modulating cholesterol homeostasis has yielded results that show that chronic use of cholesterol-lowering drugs, such as the statins, is associated with a lower incidence of AD. In APP genetically modified mice, cholesterol-lowering drugs have been shown to reduce overall pathology. While the mechanism is poorly understood it appears that cholesterol-lowering drugs have a direct effect on APP processing.  is an Alzheimer's disease drug which has received widespread approval. It is a non-competitive N-methyl-D-aspartate (NMDA) channel blocker. By binding to the NMDA receptor with a higher affinity than Mg2+ ions, memantine is able to inhibit the prolonged influx of Ca2+ ions, particularly from extrasynaptic receptors, which forms the basis of neuronal excitotoxicity. It is an option for the management of those with moderate to severe Alzheimer's disease (modest effect). The study showed that 20 mg/day improved cognition, functional ability and behavioural symptoms.  is a candidate drug for the treatment of Alzheimer's disease. It is an arginase inhibitor which readily crosses the blood brain barrier, and reduces arginine loss in the brain. Amyloid beta deposition is associated with L-arginine deprivation and neurodegeneration. Mice treated with Norvaline display improved spatial memory, increased neuroplasticity-related proteins, and decrease in amyloid beta. 
Imaging compounds, notably Pittsburgh compound B, (6-OH-BTA-1, a thioflavin), can selectively bind to amyloid beta in vitro and in vivo. This technique, combined with PET imaging, is used to image areas of plaque deposits in those with Alzheimer's. 
Post mortem or in tissue biopsies Edit
Amyloid beta can be measured semiquantitatively with immunostaining, which also allows one to determine location. Amyloid beta may be primarily vascular, as in cerebral amyloid angiopathy, or in amyloid plaques in white matter. 
One sensitive method is ELISA which is an immunosorbent assay which utilizes a pair of antibodies that recognize amyloid beta.  
Atomic force microscopy, which can visualize nanoscale molecular surfaces, can be used to determine the aggregation state of amyloid beta in vitro. 
Vibrational microspectroscopy is a label-free method that measures the vibration of molecules in tissue samples.  Amyloid proteins like Aβ can be detected with this technique because of their high content of β-sheet structures.  Recently, the formation of Aβ fibrils was resolved in different plaque-types in Alzheimer's disease, indicating that plaques transit different stages in their development. 
Dual polarisation interferometry is an optical technique which can measure early stages of aggregation by measuring the molecular size and densities as the fibrils elongate.   These aggregate processes can also be studied on lipid bilayer constructs. 
11.4F: Antimicrobial Peptides - Biology
The virtual colony count (VCC) microbiological assay has been used for over a decade to measure the effect of antimicrobial peptides such as defensins and LL-37 against a variety of bacteria. ( Ericksen et al. , 2005 Zhao et al. , 2013 ). It infers antimicrobial activity based on the quantitative growth kinetics of 200 μL batch cultures of bacteria grown in 96-well plates using a method of enumeration of viable cells ( Brewster, 2003 ) mathematically identical to the method of enumeration of amplicons utilized by quantitative real-time PCR ( Heid et al. , 1996 ). The originally published plate configuration included a ring of 36 wells containing uninoculated Mueller Hinton Broth (MHB) capable of detecting cross-contamination ( Ericksen, 2014b ). There was evidence that bacteria might form clumps and biofilms during the assay, including scatter detectable by the plate reader and the presence of ubiquitous macroscopic clumping in tryptic soy broth. 10 μL samples of cross-contamination control wells that had become turbid after VCC experiments were Gram-stained, revealing few clumps. Apparently, most clumps were not retained on the glass during the Gram stain procedure ( Gram, 1884 ), whether fixed to the slide by heat or methanol. The application of lactophenol cotton blue, ordinarily used to visualize fungi by staining cell wall polysaccharides such as chitin, revealed circles and rings consistent with the caramelized residue of polysaccharides, which presumably included capsular polysaccharides and slime secreted concomitantly with clump and biofilm formation. These dark blue circles and rings could be consistent either with a heterogeneous subpopulation of E. coli or with slight contamination with a second strain.
Materials and methods Virtual colony count
The VCC assay was conducted using the 36 edge wells to detect contamination as originally described ( Ericksen et al. , 2005 ), except a rectangular piece of Parafilm M (6 × 0.25 squares) was wrapped around the 96-well plate before the start of the 2-hour and 12-hour plate reader runs. Parafilm strips remained almost entirely intact and in place throughout the 12-hour run at 37ଌ and resulted in the complete absence of dust large enough to be visible using an Olympus 8Z61 crystallographic microscope on the ledge between the 96 wells and the edge of the plate, except for a single speck in one experiment observed near a crack in the Parafilm. Parafilm also prevented the visible decrease in edge well volume due to evaporation that originally necessitated excluding these wells from the experimental portion of the assay ( Ericksen et al. , 2005 ). This evaporation also caused a slight progressive increase in optical density to a maximum ΔOD 650 of 0.004 among the edge wells over the course of the 12-hour experiment as the Mueller Hinton Broth became more concentrated. This evaporation was too slight to affect experimental (inoculated) wells measurably or affect the linearity of the calibration curve. 10 µL samples of edge wells were added to droplets of sterile water or media and spread on Mueller Hinton Agar, Tryptic Soy Agar, and Sabouraud’s Agar plates. Colonies were analyzed by morphology, wet mounts, Gram stains, and biochemical analysis using Becton Dickinson Enteropluri Product Number 261185.
Figure 1. Quantitative growth kinetics (QGK) and the lactophenol cotton blue Gram stain.
Lactophenol Cotton Blue Gram Stain Procedure. Overnight steps allowed for equilibration to the ambient humidity during summer months in the IHV building at UMB, which ranged from 40%. Water content and temperature may be important factors for the caramelization process to be quantitatively reproducible.
Glass slides were scrubbed with PCMX hand soap using a pipe cleaner. 10 µL of cells sampled from 96-well plates after VCC assays using twice-concentrated MHB in the outgrowth step were added to the slides and equilibrated to ambient humidity overnight. The slides were heat-fixed by placing the sample at the point in space at the upper tip of the inner blue flame of a Bunsen burner three times for one second each, removing the slide for one second in between ( Figure 1 ). Ambient relative humidity was 40%. The slides were stained with Fluka Analytical Gram Staining Kit Product Number 77730 and again equilibrated to ambient humidity overnight in a vertical position. Becton Dickinson Lactophenol Cotton Blue Stain Droppers Product Number 261188 were applied to the Gram stained sample and digital images were captured using an Amscope light microscope at 160×, 400× and 1600× magnification and Toupview software. The Adobe Photoshop thresholding function was applied to the 400× digital images using a threshold of 100. Black pixels were enumerated using the histogram function.
Results Clumps were observed in E. coli cultures and in open cuvettes
Macroscopic clumps were observed in 25 mL TSB batch cultures of E. coli ATCC ® 25922™ grown at 37ଌ in early exponential phase to an expected optical density at 650 nm (OD 650) of approximately 0.3. A 1 mL uncovered sample placed in a cuvette and cooled to room temperature rapidly formed small macroscopic clumps (up to about 1 mm in diameter), some of which exhibited motility, swimming in a synchronized wave downward to form a single large macroscopic clump (up to 1 cm long, equal to the cuvette width) at the base of the cuvette. OD 650 plummeted up to 2% per minute, reaching equilibrium after a 10% decrease when placed in a room temperature HPLC detector, as cells in suspension joined the clump beneath the light path. The optical density readings declined so rapidly that only the first two digits of the four reported by the Waters 600 detector could be recorded. Observing cuvettes containing such clumps, it was apparent that cohesion, rather than adhesion, was more important, since the clumps moved downward from one corner to the other corner of the cuvette as it was rotated by hand.
Remediation of clumping and use of an open cuvette as a biosensor
Macroscopic clumping in the batch culture or cuvette outside the detector was no longer observed after four changes: 1. using a small HEPA-filtered air purifier, 2. replacing in-house deionized Milli-Q water with purchased molecular biology grade water, 3. replacing 2×MHB prepared and autoclaved in-house using reusable jars with Teknova 2× cation-adjusted MHB, and 4. filter-sterilizing phosphate buffers made near the portable air purifier, rather than autoclaving in reusable jars. Even after these remediation measures, uncovered 1 mL samples placed in the detector for 2 hours formed a macroscopic clump at the base of the cuvette accompanied by a decrease in optical density, suggesting that at least one clumping environmental factor (CEF) was concentrated by the fan and filter within detector acting as a dust trap. Thus, 1 mL samples of E. coli ATCC ® 25922™ served as biosensors for CEFs, and the detector served as a biosensor positive control.
E. coli cells were also motile on plates
Corner-seeking motility of E. coli ATCC ® 25922™ was also observed on MH agar plates wrapped in Parafilm and incubated at room temperature for 2𠄳 weeks, as indicated by the formation of a
1 cm-wide confluent ring around the entire edge of the plate, even though confluent areas and single colonies that originally appeared after 1𠄲 days were separate from the edge.
Cell clumping accompanied cross-contamination in VCC edge wells
The UMB VCC procedure was sensitive to cross-contamination in the 36 uninoculated edge wells, possibly indicating that clumping affects the particle size distribution and adhesive properties of the cells, which in turn promotes aerosol formation during pipetting ( Ericksen, 2014b ). Figure 2 depicts cells sampled from a cross-contaminated edge well after storage at 4ଌ. The UCLA VCC method, with cells in 10 µL pipetted beneath 90 µL rather than a 50 µL suspension added to 50 µL as droplets from above, ( Welkos et al. , 2011 ) minimizes the probability of cross-contamination and is a safer and more effective method of transferring bacteria such as the hazardous BSL-3 pathogen Bacillus anthracis .
Figure 2. Blue Gram stain and thresholding results at 400× magnification.
A : Blue rings indicate the polysaccharide residue of clumps of cells presumably washed from the slides during the Gram stain procedure. These polysaccharides were invisible when inspected after Gram staining and before application of lactophenol cotton blue. Other experiments produced smaller dark blue circles rather than rings. B : Thresholding results. A large majority of black pixels are contained within the polysaccharide rings.
The blue Gram stain reveals polysaccharides that are invisible after Gram staining alone
The lactophenol cotton blue Gram stain (BGS) revealed ubiquitous circular or ring-shaped structures that stained dark blue ( Figure 2A ). All cells stained light blue because all cells are glycosylated and concentrate polysaccharides from the media as part of their metabolism. Rare regions of indistinct blue staining were also observed, probably resulting from starch and other polysaccharides present in MHB, suggesting that the intensity of blue staining could also arise from starch and other carbohydrates with the capsular polysaccharides. MHB contains 1.5 g/L starch, plus a variety of other carbohydrates contained in beef extract. Carbohydrates, which must have included Maillard reaction ( Maillard, 1912 ) and caramelization products, adhered to the glass in the intense heat of the fixation steps and endured on the slide throughout the Gram stain procedure. These polysaccharide residues had been invisible when these same slides were observed after Gram staining and before application of lactophenol cotton blue. The intensity of dark blue staining suggests copious capsule and slime formation.
Polysaccharide staining can be readily quantified by thresholding
Applying the thresholding technique using a threshold of 100 differentiated the dark from the light staining with little apparent background noise ( Figure 2B ). Thresholding of BGS images captured at 160× and 1600× magnification ( Figure 3 ) are also possible using the Amscope microscope. However, pixelation could add imprecision at 160× and the large size of clumps would increase variability from field to field at 1600×. TSB or MHB cultures of E. coli ATCC ® 43827™ (ML-35) produced no macroscopic clumps under any conditions in several experiments conducted in 2013 and 2014, indicating that the observed clumping is strain-dependent.
Figure 3. Blue Gram stain results at 160×, 400× and 1600× magnification.
A – C : 160×. D – F : 400×. G – I : 1600×. Cells were sampled from the edge wells of a different virtual colony count experiment than Figure 2 .
VCC cross-contamination is ordinarily a rare event
The history of hundreds of VCC experiments at UMB between 2003 and 2014 ( Ericksen et al. , 2005 Pazgier et al. , 2012 Rajabi et al. , 2012 Wei et al. , 2009 Wei et al. , 2010 Wu et al. , 2005 Wu et al. , 2007 Xie et al. , 2005a Xie et al. , 2005b Zhao et al. , 2012 Zhao et al. , 2013 Zou et al. , 2008 ) clearly shows that edge wells are almost always clear, not turbid, after the 12h outgrowth phase of VCC experiments. In a 1-month period in August and September 2013, 13 quadruplicate calibration experiments were conducted using the same pipetting technique as the sextuplicate calibration experiments in the original VCC publication ( Ericksen et al. , 2005 ). However, in the 2013 experiments, four, rather than six, calibration curves were confined to 32 internal wells (C3-F10). These experiments used the rich media MHB, TSB or slight variations thereof. The external 64 wells (rows A, B, G and H and columns 1, 2, 11 and 12) contained two rings of contamination control wells rather than the single ring of 36 wells originally used. In these experiments conducted just outside a biosafety cabinet used for VCC experiments, none of the 832 contamination control wells turned turbid after the 12h incubation. Assuming clumping is caused by an environmental factor, these experiments strongly suggest that CEFs present in the laboratory environment are overwhelmingly non-culturable in rich media such as MHB or TSB. An alternate explanation of infrequent cell clumping and rare paradoxical points is that bacterial cells have a mechanism to induce clumping and biofilm formation infrequently and constitutively even in the absence of any causative agent or contaminant. If cell clumping is caused by a contaminant, several possible sources are present in the laboratory environment. In addition to viable contamination, unculturable bacteria could exert an influence upon rapidly growing E. coli cells. Furthermore, nucleic acids are known to cause cells to coalesce into clumps over a broad size distribution in both bacterial and mammalian cell culture. Airborne CEFs smaller than a bacterial cell could pass through the HEPA filters with little or no resistance, meaning that these molecules could have affected experiments conducted both inside and outside biosafety cabinets. Measures such as trypsinization, treatment with other proteases, and treatment with nucleases such as benzonase are commonly employed to reduce or eliminate clumping ( Kruse & Patterson, 1973 ). For the same purpose, shear was employed in VCC calibration curves by placing pipette tips in contact with the cross-sectional corner of each well when pipetting up and down 15 times to mix ( Ericksen, 2014b ), although growth curves showed evidence of clumps large enough to produce measurable differences in optical density that preceded exponential growth. Clumping had no effect on the linearity of the calibration curve, possibly indicating that a small fraction of cells routinely grow as clumps and biofilms in the absence of antimicrobial agents.
Discussion Clump formation could lead to persisters that are resistant to antimicrobial peptides
The presence of polysaccharides associated with E. coli ATCC ® 25922™ cohesion suggests that in the conditions studied at UMB, this strain employs clumping, possibly as a defense mechanism. Forming a clump surrounded by polysaccharides could contribute to resistance to antimicrobial lectins such as defensins ( Wang et al. , 2003 ) that would be bound and inhibited at the surface, limiting further inward diffusion and protecting persister cells ( Ericksen et al. , 2005 ) at the center of the clump. These survivors could contribute to the deviation from simple exponential killing ( Luria & Latarjet, 1947 ) observed throughout all VCC studies at UMB of defensin activity against E. coli . They could also explain the presence of paradoxical data points observed occasionally throughout the history of VCC experiments at UMB. For example, the defensin HNP1 at the highest concentration of 256 µg/mL caused greater survival than 128 µg/mL in the initial VCC study ( Ericksen et al. , 2005 ) MHB contains a considerable amount (1.5 g/L) of added starch. Polysaccharides in rich media could contribute to the complete inhibition of antimicrobial peptides, which is essential for VCC assays to be capable of enumerating surviving bacteria by the QGK data analysis method. Qualitative defensin lectin activity generally follows the hierarchy: glycosylated proteins > branched polysaccharides > linear polysaccharides > oligosaccharides > monosaccharides. (Lehrer, R. I., personal communication) Bacterial slime and capsules are highly branched and contain glycosylated proteins ( Wilkinson, 1958 ). If inhibition follows the same qualitative pattern as binding, bacterial capsular polysaccharides would be potent defensin inhibitors. Clump, biofilm and capsule formation may have evolved partially as resistance mechanisms to the ancient selection pressure exerted throughout the tree of life by antimicrobial peptides in the environment.
Clump formation suggests that glycosidase activity is essential for efficacy against persisters
A possible consequence of the inhibition of defensins by polysaccharides could be that therapies with lectin antimicrobial peptides as active ingredients would not be effective against clumps or biofilms in the absence of at least one other active ingredient that degrades the polysaccharide capsule, such as a glycosidase. Because polysaccharide structures in capsules and slime vary widely, as do glycosidase substrate specificities, any given enzyme might be active against only a narrow range of bacteria. In the absence of in vivo glycosidases, activity against a broad spectrum of pathogenic bacteria would therefore require an enzyme cocktail of glycosidases accompanying the lectin antimicrobial peptide or a glycosidase with unusually promiscuous substrate specificity.
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Brancatisano, F. L. et al. Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis. Biofouling 30, 435–446 (2014).
Hilpert, K., Volkmer-Engert, R., Walter, T. & Hancock, R. E. W. High-throughput generation of small antibacterial peptides with improved activity. Nat. Biotechnol. 23, 1008–1012 (2005).
Tucker, A. T. et al. Discovery of next-generation antimicrobials through bacterial self-screening of surface-displayed peptide libraries. Cell 172, 618–628.e13 (2018). This study describes a Surface Localized Antimicrobial Display platform for screening unlimited numbers of host defence peptides for a variety of functions, vastly increasing the number of known active peptide sequences.
Lee, E. Y., Lee, M. W., Fulan, B. M., Ferguson, A. L. & Wong, G. C. L. What can machine learning do for antimicrobial peptides, and what can antimicrobial peptides do for machine learning? Interface Focus 7, 20160153 (2017).
Etayash, H., Pletzer, D., Kumar, P., Straus, S. K. & Hancock, R. E. W. Cyclic derivative of host-defense peptide IDR-1018 improves proteolytic stability, suppresses inflammation, and enhances in vivo activity. J. Med. Chem. 63, 9228–9236 (2020).
Mourtada, R. et al. Design of stapled antimicrobial peptides that are stable, nontoxic and kill antibiotic-resistant bacteria in mice. Nat. Biotechnol. 37, 1186–1197 (2019). This study shows that stapled peptide derivatives are more effective and less toxic than naturally occurring peptides in a clinically relevant murine model of sepsis.
Luther, A. et al. Chimeric peptidomimetic antibiotics against Gram-negative bacteria. Nature 576, 452–458 (2019).
Carmona-Ribeiro, A. M. & Dias de Melo Carrasco, L. Novel formulations for antimicrobial peptides. Int. J. Mol. Sci. 15, 18040–18083 (2014).
Haney, E. F. et al. Computer-aided discovery of peptides that specifically attack bacterial biofilms. Sci. Rep. 8, 1871 (2018). This study describes a method for screening peptide libraries for antibiofilm activity and developing quantitative structure–activity relationship models for further optimization of screened peptides.
Xu, L. et al. Conversion of broad-spectrum antimicrobial peptides into species-specific antimicrobials capable of precisely targeting pathogenic bacteria. Sci. Rep. 10, 944 (2020).
Lemon, D. J. et al. Construction of a genetically modified T7Select phage system to express the antimicrobial peptide 1018. J. Microbiol. 57, 532–538 (2019).
Haney, E. F., Trimble, M. J., Cheng, J. T., Vallé, Q. & Hancock, R. E. W. Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides. Biomolecules 8, 29 (2018).
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Wang, Z., de la Fuente-Núñez, C., Shen, Y., Haapasalo, M. & Hancock, R. E. W. Treatment of oral multispecies biofilms by an anti-biofilm peptide. PLoS ONE 10, e0132512 (2015).
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Huang, X. et al. Effect of long-term exposure to peptides on mono- and multispecies biofilms in dentinal tubules. J. Endod. 45, 1522–1528 (2019).
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Starr, C. G. et al. Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria. Proc. Natl Acad. Sci. USA 117, 8437–8448 (2020).
11.4F: Antimicrobial Peptides - Biology
It has been reported that proteolytic enzymes are upregulated in BM after conditioning for transplantation and, by removal of three N-terminal amino acids from stromal derived factor-1 (SDF-1), abrogate its chemotactic activity, even if the SDF-1 peptide remains detectable by ELISA (Cancer Res. 201070:3402). We also reported that cationic antimicrobial peptides (CAMPs), such as complement cascade C3 protein cleavage fragment C3a, b2-defensin, and cathelicidin (LL-37) (Leukemia 2012, 26, 736), potently enhance the migration and homing responses of HSPCs to a low SDF-1 gradient, which is critical for retention of SDF-1 chemotactic activity in the highly proteolytic microenvironment of BM induced by conditioning for transplantation. This priming effect depends on incorporation of the CXCR4 receptor into membrane lipid rafts, a phenomenon that allows for more efficient interaction of the CXCR4 receptor with downstream signaling proteins (Blood 2005101:3784). However, we were aware that inhibition of lipid raft formation by b-methylcyclodextrin inhibits only ∼50% of the CAMP priming effect, which strongly indicates the involvement of other mechanisms. Interestingly, it was reported recently that LL-37, by involving pannexin channels, enhances the release of ATP from cells (J Biol. Chem. 2008263:30471) that had been described to be an autocrine chemottractant secreted at the leading edge of migrating macrophages and neutrophils (Science 2006314:1792). Exogenous ATP has also been reported in Tranwell assays to chemoattract HSPCs (Blood 2004104:1662).
We hypothesized that HSPCs, like monocytes and neutrophils, release at their leading edge ATP, and respond to this autocrine chemoattractant. Thus, this autocrine ATP migration-enhancing phenomenon could provide not only new insight into mechanisms regulating the migration of HSPCs but also better explain the priming effect of CAMPs to a low or decreasing SDF-1 gradient.
First, we tested the responsiveness of murine and human BM-, mobilized peripheral blood (mPB)-, and human umbilical cord blood (UCB)-derived HSPCs to different extracellular nucleotides including ATP by performing i) Transwell migration assays, ii) MAPKp42/44 and AKT phosphorylation studies, and iii) RQ-PCR expression of different purinergic receptors on normal HSPCs. The priming effect of CAMPs on autocrine secretion of ATP by HSPCS was studied in functional chemotaxis assays by employing the broad-spectrum ATP receptor antagonist suramin and apyrase, an enzyme that degrades extracellular ATP.
We noticed that for all the nucleotides tested (ATP, UTP, GTP, TTP, and CTP), ATP has the strongest chemotactic activity against murine and human BM-derived HSPCs, which correlated with the phosphorylation of MAPKp42/44 and AKT. In contrast to BM-derived HSPCs, the chemotactic effect of ATP and its induction of signaling were significantly attenuated for mPB- and UCB-derived HSPCs, which suggests a desensitization of purinergic surface receptors by free extracellular nucleotides circulating in blood plasma. Most importantly, we found that exposure of HSPCs to suramin or apyrase significantly diminished (by ∼50%) the priming effect of CAMPs in enhancing the responsiveness of these cells to a low SDF-1 gradient. Finally, we found that both ATP and CAMPs are secreted from irradiated BM stromal cells, which provides a heretofore underappreciated homing signal for HSPCs.
We show that ATP secreted both as a paracrine factor by BM stromal cells during conditioning for transplantation and by migrating HSPCs at their leading edge enhances migration and homing of HSPCs to BM niches. Most importantly, our data demonstrates, for first time, the involvement of an autocrine ATP–purinergic receptor migration regulatory loop, which better explains the pro-migratory priming effect seen after exposure of HSPCs to CAMPs. Since CAMPs, which enhance migration of HSPCs to a low SDF-1 gradient are upregulated like ATP in irradiated BM, all these factors together orchestrate the responsiveness of HSPCs to a decreasing SDF-1 gradient in the proteolytic microenvironment of BM conditioned for transplantation. These mechanisms should be further explored to improve the homing of HSPCs after transplantation, and we are currently testing this possibility in appropriate animal models.
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