Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A

Thennarasu, Sathiah; Lee, Dong Kuk; Poon, Alan; Kawulka, Karen E.; Vederas, John C.; Ramamoorthy, Ayyalusamy

doi:10.1016/j.chemphyslip.2005.06.003

Cited by 121 publications

(91 citation statements)

References 47 publications

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“…The mechanisms of action of most circular bacteriocins have not yet been determined, but for two of the most studied cases, enterocin AS-48 and carnocyclin A, it was previously shown that the bacteriocins can permeabilize liposomes and/or lipid bilayers (21,22). Also, previous studies of the circular bacteriocins gassericin A and subtilosin A indicated that the bacteriocins do not require a target receptor (28,55). Thus, it has been suggested that circular bacteriocins in general exert their activity independently of any target molecule in the cell membrane of sensitive cells.…”

Section: Discussionmentioning

confidence: 99%

The Maltose ABC Transporter in Lactococcus lactis Facilitates High-Level Sensitivity to the Circular Bacteriocin Garvicin ML

Gabrielsen

Brede

Hernández

et al. 2012

Antimicrob Agents Chemother

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ABSTRACTWe generated and characterized a series of spontaneous mutants ofLactococcus lactisIL1403 with average 6- to 11-fold-lowered sensitivities to the circular bacteriocin garvicin ML (GarML). Carbohydrate fermentation assays highlighted changes in carbohydrate metabolism, specifically loss of the ability to metabolize starch and maltose, in these mutants. PCR and sequencing showed that a 13.5-kb chromosomal deletion encompassing 12 open reading frames, mainly involved in starch and maltose utilization, had spontaneously occurred in the GarML-resistant mutants. Growth experiments revealed a correlation between sensitivity to GarML and carbon catabolite repression (CCR); i.e., sensitivity to GarML increased significantly when wild-type cells were grown on maltose and galactose as sole carbohydrates, an effect which was alleviated by the presence of glucose. Among the genes deleted in the mutants weremalEFG, which encode a CCR-regulated membrane-bound maltose ABC transporter. The complementation of mutants with these three genes recovered normal sensitivity to the bacteriocin, suggesting an essential role of the maltose ABC transporter in the antimicrobial activity of GarML. This notion was supported by the fact that the level of sensitivity to GarML was dose dependent, increasing with higher expression levels ofmalEFGover a 50-fold range. To our knowledge, this is the first time a specific protein complex has been demonstrated to be involved in sensitivity to a circular bacteriocin.

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Section: Discussionmentioning

confidence: 99%

The Maltose ABC Transporter in Lactococcus lactis Facilitates High-Level Sensitivity to the Circular Bacteriocin Garvicin ML

Gabrielsen

Brede

Hernández

et al. 2012

Antimicrob Agents Chemother

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“…The subtilosin A-induced dye leakage experiment showed that the leakage was observed at a peptide/lipid ratio of 1:6 from liposomes that mimic the lipid composition of bacterial membrane but not from that of mammalian membrane (31), which is consistent with the very low (if any) hemolytic activity of subtilosin A. How does the singleamino-acid substitution confer hemolytic activity?…”

Section: Discussionmentioning

confidence: 65%

“…was slightly buried in the hydrophobic membrane bilayers and that the positively charged K2 residue interacts with the negatively charged lipid head group (31). The negatively charged edge containing D16, D21, and E23 was positioned far from the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…The aforementioned dye leakage experiment showed that the membrane leakage induced by subtilosin A occurs at peptide concentrations higher than those required for bactericidal activity (31), suggesting that the bactericidal effect of subtilosin A is caused by the secondary effect, e.g., activation of autolysins or apoptosis, which is brought about by interaction of subtilosin A with the membrane. Nisin has been known to cause membrane permeabilization, but a recent study showed that the lethal action of nisin is due to processes following membrane permeabilization, which include accelerated cell division and cell envelope inhibition (8).…”

Section: Discussionmentioning

confidence: 99%

“…A study of subtilosin A with model phospholipid bilayers showed that subtilosin A interacts with the lipid head group region of the membrane and at least the W34-containing edge tilts into the phospholipid bilayers but that the negatively charged edge (D16/D21/E23) is exposed outside the membrane (31). As subtilosin A causes membrane permeabilization only at concentrations higher than those effective in vivo, its mechanism may require a receptor molecule in the target cell.…”

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confidence: 99%

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Isolation of a Variant of Subtilosin A with Hemolytic Activity

et al. 2009

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Bacillus subtilis produces an anionic bacteriocin called subtilosin A that possesses antibacterial activity against certain gram-positive bacteria. In this study, we uncovered a hemolytic mutant of B. subtilis that produces an altered form of subtilosin A. The mutant bacteriocin, named subtilosin A1, has a replacement of threonine at position 6 with isoleucine. In addition to the hemolytic activity, subtilosin A1 was found to exhibit enhanced antimicrobial activity against specific bacterial strains. The B. subtilis albB mutant that does not produce a putative immunity peptide was more sensitive to both subtilosin A and subtilosin A1. A spontaneous suppressor mutation of albB that restored resistance to subtilosin A and subtilosin A1 was obtained. The sbr (subtilosin resistance) mutation conferring the resistance is not linked to the sboA-alb locus. The sbr mutation does not increase the resistance of B. subtilis to other cell envelope-targeted antimicrobial agents, indicating that the mutation specifically confers the resistance to subtilosins. The findings suggest possible bioengineering approaches for obtaining anionic bacteriocins with enhanced and/or altered bactericidal activity. Furthermore, future identification of the subtilosin-resistant mutation could provide insights into the mechanism of subtilosin A activity.Bacteria produce a variety of secondary metabolites, such as antibiotics and bacteriocins. Bacteriocins are proteinaceous antimicrobial substances that are often distinguished from traditional antibiotics by their narrow range of activity against closely related bacteria. However, bacteriocins produced by gram-positive bacteria show a relatively broader spectrum than those from gram-negative bacteria (reviewed in references 14 and 22). Various gram-positive bacteria produce small bacteriocins that are ribosomally synthesized peptides but that are often heavily modified during posttranslational processing. The target of these bacteriocins is primarily the cytoplasmic membrane, where they create pores through which ions can pass, leading eventually to cell death.Subtilosin A is a 35-amino-acid bacteriocin originally isolated from Bacillus subtilis by Babasaki et al. (2) but was also found in Bacillus atrophaeus (26) and Bacillus amyloliquefaciens isolated from a dairy product (29). It is a unique macrocyclic bacteriocin in part due to its having an anionic nature unlike many other membrane-targeting cationic bacteriocins. Subtilosin A is formed from its precursor by proteolytic cleavage of the N-terminal leader peptide and cyclization through covalent linkage between the N-terminal asparagine and the C-terminal glycine. Structure analysis of subtilosin A by 1 H nuclear magnetic resonance (NMR) spectrometry suggested the formation of thioether bonds between Cys4/Phe31, Cys7/ Thr28, and Cys13/Phe22, but the actual sites of connection were unknown (15). Subsequent isotopic labeling and multidimensional NMR analysis demonstrated unprecedented crosslinking between sulfurs of the cysteines and the ␣ ca...

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