Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance

Abstract

The increase of infections caused by multidrug-resistant bacteria, together with the loss of effectiveness of currently available antibiotics, represents one of the most serious threats to public health worldwide. The loss of human lives and the economic costs associated to the problem of the dissemination of antibiotic resistance require immediate action. Bacteria, known by their great genetic plasticity, are capable not only of mutating their genes to adapt to disturbances and environmental changes but also of acquiring new genes that allow them to survive in hostile environments, such as in the presence of antibiotics. One of the major mechanisms responsible for the horizontal acquisition of new genes (e.g., antibiotic resistance genes) is bacterial conjugation, a process mediated by mobile genetic elements such as conjugative plasmids and integrative conjugative elements. Conjugative plasmids harboring antibiotic resistance genes can be transferred from a donor to a recipient bacterium in a process that requires physical contact. After conjugation, the recipient bacterium not only harbors the antibiotic resistance genes but it can also transfer the acquired plasmid to other bacteria, thus contributing to the spread of antibiotic resistance. Conjugative plasmids have genes that encode all the proteins necessary for the conjugation to take place, such as the type IV coupling proteins (T4CPs) present in all conjugative plasmids. Type VI coupling proteins constitute a heterogeneous family of hexameric ATPases that use energy from the ATP hydrolysis for plasmid transfer. Taking into account their essential role in bacterial conjugation, T4CPs are attractive targets for the inhibition of bacterial conjugation and, concomitantly, the limitation of antibiotic resistance dissemination. This review aims to compile present knowledge on T4CPs as a starting point for delving into their molecular structure and functioning in future studies. Likewise, the scientific literature on bacterial conjugation inhibitors has been reviewed here, in an attempt to elucidate the possibility of designing T4CP-inhibitors as a potential solution to the dissemination of multidrug-resistant bacteria.

Keywords: antibiotic resistance; bacterial conjugation; coupling proteins; drug discovery; novel conjugation inhibitors.

FIGURE 1

Molecular architecture of the T4ASS showing the location of the T4CP. Different components of the pilus, OMCC, IMC, and the ATPase energy center are shown. To show equivalence between VirB/VirD4 and R388 systems, proteins corresponding to R388 are shown in brackets. Arrows represent dynamic interactions of VirB11 protein through the conjugative process. VirD4 is highlighted in green. Red boxes highlight the proteins selected as targets for drug discovery (VirB8 and VirB11). OMCC, outer membrane core complex; IMC, inner membrane complex. The schematic representation is based on the structure described by Redzej et al. (2017). The image has been adapted from Waksman (2019).

FIGURE 2

The conjugative process. Conjugative plasmids encode all the elements necessary for conjugation, while mobilizable plasmids lack a cognate T4SS and most of them also lack the T4CP. The conjugative process can be divided into five main steps: (A) Assembly of the components. (B) Formation of the pre-initiation complex. The T4CP performs the reception of the relaxosome to the secretion channel. This interaction triggers the hexamerization of the T4CP, the ATP hydrolysis by T4CP and VirB11, and finally the activation of the secretion channel. Both conjugative and mobilizable plasmids employ the T4CP of a conjugative plasmid, except for the few mobilizable plasmids that encode their cognate T4CP. (C) Activation of the conjugative process and processing of the substrate. When the pilus recognizes a receptor bacterium, a signal is transmitted to the relaxosome and DNA is processed. Each relaxase accomplishes the processing of its cognate plasmid through the specific recognition of the oriT sequence. (D) Transfer of the substrate through the T4SS. This step is always accomplished via the T4SS encoded in the conjugative plasmid. The question mark indicates that many questions remain open. (E) Substrate reception and end of conjugation. Once the substrate enters the recipient bacterium, the ssDNA is recircularized and replicated. Color code: oriT, black; relaxase, red; T4CP, green; T4SS and pilus, blue; donor bacterium, orange; receptor bacterium, purple.

FIGURE 3

Classification of T4CPs according to their molecular architecture. Each line represents one of the five subfamilies. Examples of each family are shown in the second (molecular architecture) and third (protein name and sequence length) column. Dark green, transmembrane helices; light green, nucleotide-binding domain; white lined squares, C-terminal domain. MP?, membrane protein postulated to interact with the TMD-less T4CP to perform its in vivo activity.

FIGURE 4

TrwB_R388 structure. (A) Primary structure. The location of the domains along the sequence is shown. (B) Diagram of TrwBΔN70 monomer. The different domains are colored as in panel (A). The TMD (green), not solved in the crystal structure, is represented as two cylinders. Regarding β9 and β10 strands (orange), only their structurally solved parts are shown (PDB: 19E6). (C) Representation of four monomers (each of them in a different color) with in silico modeled TMD, β9, and β10 strands (gray). Panel (C) adapted from Gomis-Rüth et al. (2002a).

FIGURE 5

(A) TrwBΔN70 and TrwB_R388 hexamers. 1 and 3 represent the side and top view of TrwBΔN70, respectively, solved by X-ray diffraction. 2 and 4 represent the side and top view of TrwB_R388, respectively, obtained by electron microscopy. Scale bar: 50 Å. Figure adapted from Hormaeche et al. (2002). (B) Representation of the possible functional oligomeric states of T4CPs. It has been described that T4CPs interact with the T4SS as dimers (Redzej et al., 2017). As their functional structure is postulated to be a hexamer, they could perform their role as (i) homohexamers, composed of six T4CP subunits; or (ii) heterohexamers, composed of four T4CP subunits and two VirB4 subunits.

FIGURE 6

(A) 3D structure of VirB8 from Brucella suis (cyan) in complex with inhibitor B8I-1 (2-(butylamino)-8-quinolinol, magenta) (PDB ID 4AKY). (B) Detail of key residues at the binding site from PDB ID 4AKY. (C) 3D structure of TraE_pKM101 from Escherichia coli (plum) in complex with inhibitor 239852 (orange) bound at the groove (PDB ID 5WIP) and superimposed to the 3D structure of E. coli TraE_pKM101 (also in plum) in complex with inhibitor 105055 (yellow) bound to the α-helical region (PDB ID 5WIO). (D) Detail of key residues at the binding site from PDB ID 5WIP. (E) Detail of key residues at the binding site from PDB ID 5WIO.