Abstract:Tripartite efflux
pumps and the related type 1 secretion systems
(T1SSs) in Gram-negative organisms are diverse in function, energization,
and structural organization. They form continuous conduits spanning
both the inner and the outer membrane and are composed of three principal
components—the energized inner membrane transporters (belonging
to ABC, RND, and MFS families), the outer membrane factor channel-like
proteins, and linking the two, the periplasmic adaptor proteins (PAPs),
also known as the membrane … Show more
“…4a ; Supplementary Table 1b ; Supplementary Note 3 ). This was unexpected, since the TMDs of the AcrB protomers are known thus far to exclusively facilitate the transport of protons via the proton relay residues 3 , 15 . Fig.…”
Section: Resultsmentioning
confidence: 99%
“…These bacteria are intrinsically resistant against cytotoxic substances due to the synergistic action of the IM and OM and a network of multidrug efflux systems 1 . Various efflux pumps with broad overlapping drug preferences sequester and transport drugs across the IM to supply the highly polyspecific tripartite resistance–nodulation–cell division (RND) efflux pump systems with compounds to be extruded across the OM barrier 2 , 3 . In clinical Gram-negative strains, RND efflux pumps are often upregulated and contribute to the overall multidrug resistance phenotype, leaving infections untreatable with our current arsenal of antibiotics 4 .…”
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
“…4a ; Supplementary Table 1b ; Supplementary Note 3 ). This was unexpected, since the TMDs of the AcrB protomers are known thus far to exclusively facilitate the transport of protons via the proton relay residues 3 , 15 . Fig.…”
Section: Resultsmentioning
confidence: 99%
“…These bacteria are intrinsically resistant against cytotoxic substances due to the synergistic action of the IM and OM and a network of multidrug efflux systems 1 . Various efflux pumps with broad overlapping drug preferences sequester and transport drugs across the IM to supply the highly polyspecific tripartite resistance–nodulation–cell division (RND) efflux pump systems with compounds to be extruded across the OM barrier 2 , 3 . In clinical Gram-negative strains, RND efflux pumps are often upregulated and contribute to the overall multidrug resistance phenotype, leaving infections untreatable with our current arsenal of antibiotics 4 .…”
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
“…Before discussing the RND efflux pumps from different Gram-negative pathogens, we will briefly summarize our current knowledge of arguably the most studied RND pump called AcrB, from Escherichia coli (AcrB-Ec). More elaborate and detailed reviews regarding the structure and the mechanism of AcrB-Ec and other multidrug transporters can be read elsewhere [12,[28][29][30][31][32]. In short, the first crystal structure of an RND-type multidrug efflux pump (AcrB-Ec) was solved in 2002 [33], paving the way for concise structure-function analysis after previous meticulous biochemical analysis of this efflux pump before this crystal structure was available, e.g., [34,35].…”
Section: Structure Of Rnd-type Multidrug Efflux Pumpsmentioning
The rise in multidrug resistance (MDR) is one of the greatest threats to human health worldwide. MDR in bacterial pathogens is a major challenge in healthcare, as bacterial infections are becoming untreatable by commercially available antibiotics. One of the main causes of MDR is the over-expression of intrinsic and acquired multidrug efflux pumps, belonging to the resistance-nodulation-division (RND) superfamily, which can efflux a wide range of structurally different antibiotics. Besides over-expression, however, recent amino acid substitutions within the pumps themselves—causing an increased drug efflux efficiency—are causing additional worry. In this review, we take a closer look at clinically, environmentally and laboratory-evolved Gram-negative bacterial strains and their decreased drug sensitivity as a result of mutations directly in the RND-type pumps themselves (from Escherichia coli, Salmonella enterica, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Acinetobacter baumannii and Legionella pneumophila). We also focus on the evolution of the efflux pumps by comparing hundreds of efflux pumps to determine where conservation is concentrated and where differences in amino acids can shed light on the broad and even broadening drug recognition. Knowledge of conservation, as well as of novel gain-of-function efflux pump mutations, is essential for the development of novel antibiotics and efflux pump inhibitors.
“…However, a limited number of tripartite systems have been identified which span the entire cell envelope in Gram-negative bacteria ( Figure 1 ). In the latter case, the MFS component is connected to a periplasmic adaptor protein, which in turn is linked to TolC or to a TolC-like protein in the outer membrane to provide a continuous sealed channel for translocation of the substrate across the cell envelope ( Alav et al, 2021 ). At variance with tripartite assemblies involving ABC and RND transporters, tripartite assemblies including MFSs are still poorly characterized from a structural point of view.…”
Section: How Multidrug Resistance Efflux Pumps Of the Major Facilitator Superfamily Export Molecules: Structure And Functionmentioning
confidence: 99%
“…In Gram-negative bacteria they can form a tripartite complex spanning the entire cell envelope. Tripartite EPs are constituted by an inner membrane transporter protein connected with an outer membrane factor (OMF) via a periplasmic adaptor protein (PAP) ( Hinchliffe et al, 2013 ; Alav et al, 2021 ). MDR EPs have been identified within all major families of transporters, i.e., the Resistance Nodulation Division (RND), the ATP Binding Cassette (ABC), the Major Facilitator Superfamily (MFS), the Multidrug and Toxic Compound Extrusion (MATE), the Drug/Metabolite transporter (DMT), the Proteobacterial Antimicrobial Compound Efflux (PACE) family and the p-aminobenzoyl-glutamate Transporter (abgT) ( Henderson et al, 2021 ).…”
Bacterial pathogens are able to survive within diverse habitats. The dynamic adaptation to the surroundings depends on their ability to sense environmental variations and to respond in an appropriate manner. This involves, among others, the activation of various cell-to-cell communication strategies. The capability of the bacterial cells to rapidly and co-ordinately set up an interplay with the host cells and/or with other bacteria facilitates their survival in the new niche. Efflux pumps are ubiquitous transmembrane transporters, able to extrude a large set of different molecules. They are strongly implicated in antibiotic resistance since they are able to efficiently expel most of the clinically relevant antibiotics from the bacterial cytoplasm. Besides antibiotic resistance, multidrug efflux pumps take part in several important processes of bacterial cell physiology, including cell to cell communication, and contribute to increase the virulence potential of several bacterial pathogens. Here, we focus on the structural and functional role of multidrug efflux pumps belonging to the Major Facilitator Superfamily (MFS), the largest family of transporters, highlighting their involvement in the colonization of host cells, in virulence and in biofilm formation. We will offer an overview on how MFS multidrug transporters contribute to bacterial survival, adaptation and pathogenicity through the export of diverse molecules. This will be done by presenting the functions of several relevant MFS multidrug efflux pumps in human life-threatening bacterial pathogens as Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae, Shigella/E. coli, Acinetobacter baumannii.
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