Structural basis of triclosan resistance
Graphical abstract
Introduction
Triclosan (5-chloro-2-(2,4-dichloro-phenoxy)-phenol, TCL) is a broad-spectrum biocide (Levy, 2001, Schweizer, 2001). Currently, its usage is expanded in consumer products such as toothpaste, mouthwashes, deodorants, hand soaps, and lotions. It is also incorporated in children’s toys, cutting boards, and plastic films to wrap meat products. Although several mechanisms of resistance to TCL were reported including activating efflux activities, capturing the TCL and increasing the expression of important regulators or metabolic enzymes (Yu et al., 2010), mutation of ENR gene showed dramatic change in MIC value. When triclosan-resistant strains were isolated from laboratories and hospitals, the mutations included G93S, G93V, M159T and F203L (McMurry et al., 1998, Chen et al., 2009). The ENR encoded by the fabI gene is an enzyme critical for cell-wall synthesis of bacteria. Subsequent studies show that TCL is an inhibitor of ENR (Heath et al., 1998, Heath et al., 1999, Levy et al., 1999, Qiu et al., 1999). Because TCL is broadly used in our daily life, the resistance may cause serious global health problems (Levy, 2001, Aiello and Larson, 2003, Russell, 2004). ENR is now one of the most important drug targets (Fidock et al., 2004, Kuo et al., 2003, Wang et al., 2006) for methicillin-resistant staphylococcus aureus (MRSA) (Priyadarshi et al., 2010), Tuberculosis (TB) (Gagneux et al., 2006) and Malaria (Hall et al., 2005), three of major infectious problems of the world. Thus, the structural origin of TCL resistance should be understood to solve the resistance problems and discover new generation antibiotics for global infectious problems. Hence, it is vital to explore the structural changes of ENRs caused by mutation. Here we report the crystal structures of mutated ENRs complexed with TCL and NAD+ using X-ray crystallography along with the energetic analysis by molecular/quantum mechanical computation. We elucidate why the single G93V mutation of ENR raises serious consequences on the triclosan resistance.
Among the identified single amino-acid mutations in ENR of Escherichia coli K12 i.e., G93V, M159T and F203L, the highest level of TCL resistance occurs in G93V mutants (McMurry et al., 1998). The G93A/S/C/V substitution greatly reduces diazaborine (an anti-TB drug) binding to E. coli ENR (de Boer et al., 1999). The X-ray data for structures of ENR of E. coli bound with TCL and cofactor NAD+ are reported (Levy et al., 1999, Qiu et al., 1999). Despite various mutational analysis of ENR of E. coli (Levy, 2001, McMurry et al., 1998, Qiu et al., 1999), P. falciparum (Kapoor et al., 2004), and MRSA (Takahata et al., 2007) as well as crystal structural studies of mutant Mycobacterium tuberculosis ENR-NAD+ complexes (Oliveira et al., 2006) are already been reported, the structural data for mutant ENRs complexed with TCL and cofactor NAD+ are still lacking.
Section snippets
Protein expression and purification
The full length of E. coli ENR (residue 1-262) was cloned into the Nde I/Xho I sites of pET21b expression vector (Novagen) with a C-terminal 6 His-tag. The sequence information is (1-262a.a + LE (restriction site) + His-6) MGFLSGKRILVTGVASKLSIAYGIAQAMHREGAELAFTYQNDKLKGRVEEFAAQLGSDIVLQCDVAEDASIDTMFAELGKVWPKFDGFVHSIGFAPGDQLDGDYVNAVTREGFKIAHDISSYSFVAMAKACRSMLNPGSALLTLSYLGAERAIPNYNVMGLAKASLEANVRYMANAMGPEGVRVNAISAGPIRTLAASGIKDFRKMLAHCEAVTPIRRTVTIEDVGNSAAFLCSDLSAGISGEVVHVDGGFSIAAMNELELKLEHHHHHH. The His
Results and discussion
We have prepared the wild type (WT) and glycine 93 mutated (G93A, G93S and G93V mutants) E. coli ENRs. Inhibitory activity of TCL was assayed against the purified proteins. Compared with the activity against WT (IC50 = 150 nM), those against the Ala and Ser mutants are IC50 = 380 and 470 nM, respectively, while that against the G93V mutant is 68,500 nM. This result is well correlated with minimum inhibition concentration (MIC) of TCL against resistant strains containing the mutant ENRs (G93S: 4 times,
Conclusions
It should be noted that the substantial energy difference between WT and G93V is not due to their substantial structural differences, but due to only minimal structural changes of a rotated aromatic ring and slightly pushed conformation of a flexible loop. Therefore, this substantial energy difference arises solely from the H–π interactions; these insignificant structural changes depicting significant energy differences are not easily observable due to the unclear resolution of the H positions
Acknowledgments
We thank the staff at Pohang Light Source (PLS) for assistance with data collection and Dr. Kwang Jin Oh of KISTI for his help and discussion. This work was supported by the National Research Laboratory Program, subsidized MOST and MOICE of Korea, NRF (Global Research Lab project and National Honor Scientist Program). Computation was carried out at the KISTI Supercomputing Center (KSC-2008-K08-0002).
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These authors contributed equally to this work.