Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Avitabile, Concetta; D’Andrea, Luca Domenico; Romanelli, Alessandra

doi:10.1038/srep04293

Cited by 107 publications

(111 citation statements)

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“…It is acknowledged that most ␣-helical AMPs are unstructured in aqueous solution and adopt an ␣-helical conformation upon interaction with the bacterial membrane (40). Although unstructured in pure water, LL-37 was reported to be ␣-helical in most physiological buffers and in solutions containing the ion compositions of biological fluids (39).…”

Section: Discussionmentioning

confidence: 99%

“…S2 in the supplemental material) (34)(35)(36)(37)(38). Circular dichroism (CD) showed that both AMPs are unstructured in water, although they adopt an ␣-helical conformation in the presence of the organic solvent 2,2,2-trifluoroethanol (TFE) or upon interaction with membrane phospholipids (35,39,40). The conformation of cathelicidins in host physiological fluids remains unclear, since these fluids are incompatible with CD experiments.…”

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confidence: 96%

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Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

et al. 2015

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Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was ␣-helical and CRAMP was unstructured under our experimental conditions. By altering the ␣-helicity of LL-37 and CRAMP, we found that decreasing LL-37 ␣-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP ␣-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments. IMPORTANCEOmptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases. Bacterial proteases are key virulence factors involved in hostpathogen interactions. Omptins are outer membrane (OM) proteases commonly found in Gram-negative pathogens. They play important roles at the host-pathogen interface by processing or degrading a variety of host and bacterial proteins (1-10). Many omptins are found among pathogens of the Enterobacteriaceae family (11). The prototypical omptins are Escherichia coli OmpT and Yersinia pestis Pla, which inactivate antimicrobial peptides (AMPs) and activate plasminogen into plasmin, respectively (4, 6). Omptins are ␤-barrel proteases embedded ...

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Section: Discussionmentioning

confidence: 99%

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confidence: 96%

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

et al. 2015

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“…We measured changes in circular dichroism of the Mig1 fusion construct upon addition of PEG (Figure 1(k)) in a wavelength range known to be sensitive to transitions between ordered and intrinsically disordered states [71,72]. We also noted similar levels of disorder content in the Msn2 protein far from the Zn finger motif.…”

Section: Resultsmentioning

confidence: 71%

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Leake

2018

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High-speed single-molecule fluorescence microscopy in vivo shows that transcription factors in eukaryotes can act in oligomeric clusters mediated by molecular crowding and intrinsically disordered protein. This finding impacts on the longstanding puzzle of how transcription factors find their gene targets so efficiently in the complex, heterogeneous environment of the cell.Abbreviations CDF - cumulative distribution function; FRAP - fluorescence recovery after photobleaching; GFP - Green fluorescent protein; STORM - stochastic optical reconstruction microscopy; TF - Transcription factor; YFP - Yellow fluorescent protein

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“…[6] have been reported to exert strong anticancer effect against various types of cancers such as breast, prostrate, ovarian and skin cancers [7]. Considering the immense amount of AMP sequences deciphered till date [8] such peptide based drugs can prove out to be vital novel anticancer pharmaceuticals with minimal side effects.…”

Section: Introductionmentioning

confidence: 99%

Nisin Augments Doxorubicin Permeabilization and Ameliorates Signaling Cascade during Skin Carcinogenesis

Preet

Satish²,

ey³

et al. 2016

Transl Med (sunnyvale)

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Research ArticleOpen Access [12]. We have also recently demonstrated an additive in vivo anti-cancer effect of nisin in conjunction with a conventional chemotherapeutic; doxorubicin (DOX) against murine skin carcinogenesis [13]. The present study further delineates the underlying mechanism of nisin-DOX additive anticancer action in vivo (in a murine model) and in vitro (using HaCaT cell lines) against skin carcinogenesis. Materials and Methods MaterialAll the chemicals used in the present study were of analytical grade and were obtained from standard companies. ELISA plates, tissue culture plates (96 /8 well), culture dishes, Serological plates, Roux culture bottles, 0.2µm sterile syringe filters (non-pyrogenic). DMBA (7,12-Dimethylbenzylanhracene), TPA (12-O-tetradecanoylphorbol-13-acetate), RPMI 1640 (Rose Well Park Memorial Institute Lab no. 1640) (Hi-media, India), doxorubicin nisinand3-(4,5-dimethylthiazol2yl)-2,5-diphenyl tetrazolium bromide were procured from Sigma Aldrich.N-phenyl-1-napthylamine (NPN), bovine serum albumin (BSA), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), thiobarbituric acid (TBA), ethanol, formaldehyde, hydrogen peroxide (H 2 O 2 ), benzene, paraffin wax, xylene, trichloroacetic acid (TCA), sodium chloride (NaCl), EDTA, Folin's Reagent, sodium carbonate, copper sulphate, AbstractBackground: Multidrug resistance exhibited by cancerous cells has proved to be a big hurdle in the development of an effective anti-cancer therapy. We previously demonstrated nisin-doxorubicin adjunct therapy to exhibit strong additive anti-cancer effect against DMBA -induced murine skin carcinogenesis but the mechanism remained unexplored. Methods:The in vivo tumoricidal activity of the combination was validated in terms of animal bioassay observations while ex vivo anti-cancer effect was monitored by employing HaCat cell lines. Results:The combination was found to be additive in vivo as evidenced by larger decreases in mean tumor burden and tumor volumes. The IC 50 values of nisin and DOX alone were evaluated to be 16 µg/ml and 4 µg/ml respectively while the sub-inhibitory concentration of DOX was reduced to 2 µg/ml when nisin was used as an adjunct. Q values calculated using MTT assays indicated the combination to be synergetic than additive. The mechanism was found to involve enhanced membrane permeabilization as well as reduced expression of NF-Κb, TNF-α, TNF-β, IL-1 and IL-6. Conclusion:As combined effect of nisin and DOX even at halved concentrations was significantly higher than either drug alone, these observations might help in lowering the chemotherapeutic doses of DOX thereby decreasing its associated side effects.

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Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Cited by 107 publications

References 34 publications

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Nisin Augments Doxorubicin Permeabilization and Ameliorates Signaling Cascade during Skin Carcinogenesis

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