Inactivation of Polymyxin by Hydrolytic Mechanism

Abstract

Polymyxins are nonribosomal peptide antibiotics used as the last-resort drug for treatment of multidrug-resistant Gram-negative bacteria. However, strains that are resistant to polymyxins have emerged in many countries. Although several mechanisms for polymyxin resistance have been well described, there is little knowledge on the hydrolytic mechanism of polymyxin. Here, we identified a polymyxininactivating enzyme from Bacillus licheniformis strain DC-1 which was produced and secreted into the medium during entry into stationary phase. After purification, sequencing, and heterologous expression, we found that the alkaline protease Apr is responsible for inactivation of polymyxins. Analysis of inactivation products demonstrated that Apr cleaves polymyxin E at two peptide bonds: one is between the tripeptide side chain and the cyclic heptapeptide ring, the other between L-Thr and L-␣-␥-diaminobutyric acid (L-Dab) within the cyclic heptapeptide ring. Apr is highly conserved among several genera of Gram-positive bacteria, including Bacillus and Paenibacillus. It is noteworthy that two peptidases S8 from Gram-negative bacteria shared high levels of sequence identity with Apr. Our results indicate that polymyxin resistance may result from inactivation of antibiotics by hydrolysis. KEYWORDS Bacillus licheniformis, alkaline protease, polymyxin resistance, polymyxininactivating enzyme, polymyxins L-␣-␥-diaminobutyric acid (L-Dab) residues (Fig. 1). Due to the higher affinity between L-Dab and lipid A, polymyxins displace divalent cations and bind to lipid A molecules, resulting in destabilization of the outer membrane. The hydrophobic domains of polymyxins (including the fatty acyl chain and D-Phe/D-Leu-L-Leu) insert between the acyl chains of lipid A and then enter the periplasm via a self-promoted uptake mechanism. Subsequently, polymyxins penetrate into the phospholipid bilayer of the RESULTSPolymyxins can be inactivated by Bacillus species. Studies from several decades ago indicated that colistin-inactivating enzymes are probably produced and secreted by the producer of colistin, P. polymyxa (20)(21)(22). To identify the colistin-inactivating enzyme, the cylinder-plate diffusion method was performed to screen colistin-degrading bacteria from our laboratory stocks of Bacillus strains. Escherichiacoli strain DH5␣ was used as a colistin-sensitive indicator strain. The addition of colistin (10,000 U/ml) resulted in an FIG 1 Chemical structure and degradation pathway of colistin. The amino acid positions are numbered in accordance with the discussion in the text. The amino acid at position 6 is D-Phe in polymyxin B. Both colistin and PMB are mixtures of structurally related compounds. Fatty acid, 6-methyloctanoic acid for colistin A and PMB 1 and 6-methylheptanoic acid for colistin B and PMB 2

The History of Colistin Resistance Mechanisms in Bacteria: Progress and Challenges

“…To date, colistin degradation has not been related to colistin resistance in other organisms [ 30 ]. Recently, a polymyxin inhibitory enzyme of Bacillus licheniformis strain DC-1 has been identified [ 31 ]. It is an alkaline Apr protease, which cleaves colistin at two peptide bonds: one between the tripeptide side chain and the heptapeptide ring, and the other between l-Thr and l-Dab within the heptapeptide ring [ 31 ].…”

“…Recently, a polymyxin inhibitory enzyme of Bacillus licheniformis strain DC-1 has been identified [ 31 ]. It is an alkaline Apr protease, which cleaves colistin at two peptide bonds: one between the tripeptide side chain and the heptapeptide ring, and the other between l-Thr and l-Dab within the heptapeptide ring [ 31 ]. One of the main contributors to development of antibiotic resistance in bacteria is the acetylation of antibiotics.…”

Some Properties of Adsorptive Carbon Dressing Applicable to Questions of Postoperative Treatment of Wounds in Rats With Guerin Carcinoma

“…Многообразие биохимических механизмов антибиотикорезистентности, включает: модификацию мишени действия АБП; снижение/утрату способности мишени связываться с АБП; их инактивацию; эффлюкс АБП из микробной клетки; модуляцию проницаемости оболочки микробной клетки и резкое снижение эффективности транспорта АБП, и, наконец, способность бактерий синтезировать белки, предотвращающие связы-вание АБП с мишенью [5,6] и лежит в основе «системы быстрого реагирования» и появления новых устойчивых форм микроорганизмов [7]. Неуклонный рост лекарственно-устойчивых инфекций, высокая стоимость создания новых АБП и растущее нежелание фармацевтических компаний заниматься этим диктуют необходимость разработки новых антимикробных стратегий, одной из которых является разработка раневых покрытий, противомикробное действие которых носит неспецифический характер.…”

Mechanisms and evolution of colistin resistance in clinical Enterobacteriaceae

“…However, the production of these enzymes was not described in Gram-negative bacteria, but in the colistin-producing Gram-positive P. polymyxa var. colistinus (113), and Bacillus licheniformis (114). These hydrolytic enzymes have been found to be inactive against daptomycin and gramicidin, which are other antibiotic compounds with a peptide portion active against Gram-positive bacteria (114).…”

“…colistinus (113), and Bacillus licheniformis (114). These hydrolytic enzymes have been found to be inactive against daptomycin and gramicidin, which are other antibiotic compounds with a peptide portion active against Gram-positive bacteria (114). The ability to degrade other types of antimicrobial peptides (including human antimicrobial peptide LL-37), has not been investigated.…”