Molecular mechanisms of vancomycin resistance

Abstract

Vancomycin and related glycopeptides are drugs of last resort for the treatment of severe infections caused by Gram-positive bacteria such as Enterococcus species, Staphylococcus aureus, and Clostridium difficile. Vancomycin was long considered immune to resistance due to its bactericidal activity based on binding to the bacterial cell envelope rather than to a protein target as is the case for most antibiotics. However, two types of complex resistance mechanisms, each comprised of a multi-enzyme pathway, emerged and are now widely disseminated in pathogenic species, thus threatening the clinical efficiency of vancomycin. Vancomycin forms an intricate network of hydrogen bonds with the d-Ala-d-Ala region of Lipid II, interfering with the peptidoglycan layer maturation process. Resistance to vancomycin involves degradation of this natural precursor and its replacement with d-Ala-d-lac or d-Ala-d-Ser alternatives to which vancomycin has low affinity. Through extensive research over 30 years after the initial discovery of vancomycin resistance, remarkable progress has been made in molecular understanding of the enzymatic cascades responsible. Progress has been driven by structural studies of the key components of the resistance mechanisms which provided important molecular understanding such as, for example, the ability of this cascade to discriminate between vancomycin sensitive and resistant peptidoglycan precursors. Important structural insights have been also made into the molecular evolution of vancomycin resistance enzymes. Altogether this molecular data can accelerate inhibitor discovery and optimization efforts to reverse vancomycin resistance. Here, we overview our current understanding of this complex resistance mechanism with a focus on the structural and molecular aspects.

Keywords: antibiotic resistance; enzymes; glycopeptides; microbiology; structural biology; vancomycin.

Figure 1

(a) Crystal structure of vancomycin•di‐acetyl‐Lys‐d‐Ala‐d‐Ala (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=1FVM 8). The hydrogen bond lost if d‐Ala‐d‐Ala is modified to d‐Ala‐d‐lac is indicated by a pink star and pink dashes, while the region of steric clash if d‐Ala‐d‐Ala is modified to d‐Ala‐d‐Ser is indicated with a blue star. (b) Representative peptidoglycan synthesis pathway showing reactions catalyzed by housekeeping enzymes labeled by enzyme name in black text, while those catalyzed by vancomycin resistance enzymes labeled by enzyme name in red text. VanA and VanC ligases shown as representative for d‐Ala‐d‐lac and d‐Ala‐d‐Ser ligases, respectively. Modification of Lipid II to terminate in d‐lac or d‐Ser lowers affinity toward vancomycin. Adapted from9, 10

Figure 2

Vancomycin resistance operons organized by d‐Ala‐d‐lac and d‐Ala‐d‐Ser mechanisms, and then by the cassette name. Gene sizes not to scale. Key: black outline for gene arrow = insoluble protein in expression attempts by CSGID and no evidence in literature for soluble protein; tan green fill = purified by CSGID and/or in the literature; dark green fill = crystal structure solved (PDB codes indicated under gene arrow)

Figure 3

Crystal structures of vancomycin resistance ligases. (a) Structure of VRE VanA•Mg²⁺•ADP•phosphinate inhibitor complex (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=1E4E 34). The ω‐loop that participates in subsite 2 for binding of the carboxyl‐terminal d‐Ala is labeled and zoom in shows details of residues, particularly His244, thought to confer specificity against d‐Ala‐d‐lac. In zoom, double‐ended arrow indicates potential clash between His244 and the ester bond of d‐Ala‐d‐lac should that compound be present in the active site. (b) Structure of VanG•ADP complex (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=4FU0 37). The ω‐loop of this enzyme is shown but is in a conformation away from subsite 2

Figure 4

Crystal structure of dipeptidase VanX_A•Zn₂₊•d‐Ala‐d‐Ala complex (personal communication from C.H. Park, also PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=1R44 = apoenzyme structure45). Left = cartoon + sticks representation, zoom shows the details of d‐Ala‐d‐Ala binding and H‐bond between carbonyl oxygen of Tyr109 and the amide bond of d‐Ala‐d‐Ala. Right = surface representation showing small size of d‐Ala‐d‐Ala binding cleft

Figure 5

(a) Crystal structures of pentapeptidase VanY_B in complex with Cu²⁺ and d‐Ala‐d‐Ala (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=5ZHW 68), shown in the same orientation as VanX_A in Figure 4. Left = two views are shown, rotated 120°. d‐Ala‐d‐Ala shown in black sticks. Right = bottom view in surface representation, showing larger size of active site cleft. (b) Structure of the VanXY_C•Cu²⁺•d‐Ala‐d‐Ala complex (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=4OAK 67), shown in the same orientation as VanX_A in Figure 4. The bisubstrate selectivity loop and Leu113 in this loop, which interact with d‐Ala‐d‐Ala, are shown in red

Figure 6

Crystal structure of VanT_G racemase (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=4ECL 86). Two chains of the homodimer are shown in thin line and cartoon representation. PLP‐d‐Ser is from the structure of alanine racemase (http://bioinformatics.org/firstglance/fgij//fg.htm?mol=1L6F 89) that was superimposed on VanT_G. Asn696, which is in position to interact with d‐Ser, along with nearby residues, are highlighted in the zoom‐in detail

Figure 7

Crystal structure of VanU_G (PDB http://bioinformatics.org/firstglance/fgij//fg.htm?mol=3TYS). Two chains in the homodimer and the helix‐turn‐helix are indicated. Putative DNA binding residues in recognition helix are labeled