Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption

Sato, Yuji; Wang, Yi; Song, Yuchi; Geng, Weiming; Yan, Shaonan; Nakamura, Kiminori; Kikukawa, Takashi; Demura, Makoto; Ayabe, Tokiyoshi; Aizawa, Tomoyasu

doi:10.1007/s00726-021-03115-3

Cited by 4 publications

(7 citation statements)

References 52 publications

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“…To investigate the structural effects of crp4oxi and crp4red on microbes, we analyzed the proteins using circular dichroism (CD) spectroscopy ( Figure 7 ). Consistent with previous CD analysis reports [ 40 , 41 ], crp4oxi in 10 mM sodium phosphate buffer (PB) showed a strong negative maximum at 200 nm, and a positive maximum at 225 nm, confirming the presence of a disulfide bond-stabilized β-sheet structure [ 42 ]. The addition of 10–40% trifluoroethanol (TFE), to confirm the structural changes in the hydrophobic environment, did not alter the spectra.…”

Section: Resultssupporting

confidence: 90%

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

et al. 2023

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Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.

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Section: Resultssupporting

confidence: 90%

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

et al. 2023

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“…Finally, the contribution of the disulfide bonds to the antibacterial activity of cryptdins is sequence-dependent and species specific. Although we confirmed that removal of the disulfide bonds in Crp4 either had little functional impact or improved its antibacterial activity ( 52 , 54 , 55 , 58 ), we did not fully recapitulate Crp4 results with Crp1 and Crp14. Loss of disulfide bonds in Crp1 and Crp14, while functionally detrimental to the killing of S. aureus , has the opposite effect on their killing of E. coli , a dramatic enhancement for Crp1 and a marginal reduction for Crp14.…”

Section: Discussioncontrasting

confidence: 67%

“…Published studies give a variety of accounts for the functional ramification of disulfide bonds in human defensins, ranging from a reduction, no change, or an enhancement in bactericidal activity often in a species-specific fashion ( 72 – 75 ). Much of the previous SAR studies on cryptdins has focused on the reduced version of Crp4 ( 52 , 54 , 55 , 58 ) which, in general, has exhibited a bactericidal activity equivalent to or greater than that of its natively folded form. To better understand how the three invariant disulfide bonds in cryptdins impact their bactericidal activity, we selected three cryptdins, Crp1, Crp4, and Crp14, and synthesized their corresponding linear isoforms in which the six Cys residues are all replaced by Ala. As shown in Table 1 and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 ) ( 66 , 74 , 80 – 82 ). Crp4 stands out as the only cryptdin that has been extensively interrogated for its SAR with respect to its conserved salt bridge ( 51 , 56 ), cationicity ( 53 , 57 , 59 , 60 ), and disulfide bond pattern importance ( 52 , 54 , 55 , 58 ). Our present study with the 17 cryptdin isoforms originally discovered by Ouellette and colleagues from a single jejunal crypt ( 37 ) contributes to a better understanding of how cryptdins function at the molecular level.…”

Section: Discussionmentioning

confidence: 99%

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Mouse α-Defensins: Structural and Functional Analysis of the 17 Cryptdin Isoforms Identified from a Single Jejunal Crypt

et al. 2023

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“…There are six different isoforms of Crp (Fig. 1; Table 1) [19], and most studies to date have been conducted on Crp4 [20][21][22] and only a few on other Crps. The level of gene expression of different Crps in various positions in the small intestine differs, and their dissimilar characteristics indicate that different isoforms appear to have specific roles in the small intestine [19,[23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation

et al. 2023

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Background A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. Results To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. Conclusion In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

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Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption

Cited by 4 publications

References 52 publications

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

Mouse α-Defensins: Structural and Functional Analysis of the 17 Cryptdin Isoforms Identified from a Single Jejunal Crypt

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation

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