Bacterial Drug Efflux Systems Biology Essay

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The last of the resistance mechanisms to be identified, efflux was first described as a mechanism of resistance to tetracycline in E. coli [90]. In recent years, interest in efflux-mediated resistance in bacteria has been raised by the growing amount of data implicating efflux systems in resistance development in clinical isolates [80;99]. Biomembranes constitute efficient barriers towards hydrophilic molecules, most of which can penetrate cells only by specific inward-bound transport systems. Amphiphilic compounds, on the other hand, can easily cross biomembranes, since these are able to diffuse through both the hydrophilic and the hydrophilic domains of the bilayer. Hence, it is not surprising that mechanisms have evolved to protect cells from the invasion of amphiphilic molecules. A major mechanism in this respect is constituted by active efflux. Antibiotics are often amphiphilic ensuring their wide tissue distribution and their penetration into membrane-protected compartments. Thus, many drugs fall into this category of exogenous compounds for which efflux mechanisms are numerous and fairly active. Over the last years efflux systems have been recognized and characterized in almost all cell types, from prokaryotes and archaebacteria through fungi and higher eukaryotes [133]. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds; such pumps can be associated with multiple drug resistance (MDR). The up-regulation of efflux systems through physiological induction and spontaneous mutation can significantly lower the intracellular concentration of many antibiotics, causing an impact on clinical efficacy. Over-expression of efflux pumps can result from mutations within local repressor genes [2] or may result from activation of a regulon regulated by a global transcriptional regulator such MarA of E. coli [4].

Classes of microbial efflux pumps

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On the basis of bioenergetic and structural criteria, drug transporters can be divided into two major classes. ATP-binding cassette (ABC)-type primary drug transporters utilize the free energy of ATP hydrolysis to pump drugs out of the cell and are mostly transport proteins. Secondary drug transporters use the transmembrane electrochemical gradient of protons (proton-motive force, PMF) or sodium ions to drive the extrusion of drugs from the cell [110]. These secondary transporters can be subdivided into distinct families of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, the resistance-nodulation-cell division (RND) family, and the multidrug and toxic compound extrusion (MATE) family (Table 1, Fig. 1). These families are not solely associated with drug export but include proteins involved in the uptake of essential nutrients and ions, excretion of metabolic end products and deleterious substances, and communication between cells and the environment. They occur either as single-component transporters, or as multi-component systems containing not only cytoplasmic domains but also outer membrane channel proteins and periplasmic membrane fusion proteins.

Mechanism of transport

Superfamily

Examples

Substrate specificity

Number of aa residues

Topology (TMS)

ATP-dependent transporters

ABC

MsrA, S.aureus

LmrA, L.lactis

Specific, MDR

Variable

6 or 12

Secondary active transporters

MFS

NorA, S.aureus

MefE & PmrA, S.pneumoniae

MDR

400-600

12, 14 or 24

SMR

Qac, S.aureus

Specific, MDR

~110

4

RND

AcrAB, E.coli

MDR

≤1000

12

MATE

NorM, V.parahaemolyticus

MepA, S.aureus

MDR

~ 450

12Table 1. Classification of bacterial drug efflux pumps. ABC, ATP-binding Cassette; MDR, Multidrug resistant; TMS, transmembrane segments. Classification based on Refs. [110;133;134].

Fig. 1. Bacterial Drug Efflux Proteins (Langton et al. [77])

Pneumococcal efflux systems

Although resistance in S. pneumoniae is often related to target alterations, such as in penicillin-binding proteins (PBPs) or DNA gyrase/topoisomerase IV, efflux pumps clearly make an important contribution [14;120].

Active efflux of ciprofloxacin was documented in both wild-type and resistant pneumococci. Initially identified in 1999 [52], the PmrA pump is a homologue of NorA in Staphylococcus aureus (24% identity) and produces resistance to fluoroquinolones and dyes [109]. Disruption of pmrA in wild-type strains does not alter drug susceptibility, suggesting that the gene is not likely to be expressed in wild-type pneumococci [52].

Efflux pumps also play an important role in macrolide resistance in S. pneumoniae. Efflux is mediated by the genes of the genetic element mega (macrolide efflux genetic assembly) and related insertion elements, such as Tn1207.1 and Tn1207.3. These elements contain two adjacent genes, mef (mef(E) or mef(A)) and the closely related mel gene, encoding a proton motive force pump and a putative ATP-binding cassette transporter homolog, and are transcribed as an operon [5;50]. The Mef/Mel system represents a substrate-specific dual efflux pump mediating resistance to 14- and 15-membered macrolides but not to 16-membered macrolides, lincosamides or analogues of streptogramin B.

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Fig. 2. S. pneumoniae substrate transport, carbohydrate and glutamine metabolism, and selected categories of cell surface proteins [65].

Impact on resistance

Over-expression of a drug efflux pump alone generally does not confer high-level, clinically significant resistance to antibiotics. However, such bacteria are better equipped to survive antibiotic pressure and may develop further mutations in genes encoding the target sites of antibiotics [71]. It has been demonstrated that expression of the AcrAB efflux system in E. coli is greatest when the bacteria are stressed, e.g. growth in a nutrient-poor medium, growth to stationary phase or osmotic shock; these inhospitable conditions may be relevant to the situation within an infection [111]. Unregulated over-expression of efflux pumps is potentially disadvantageous to the bacterium as not only will toxic substrates be exported but also nutrients and metabolic intermediates may be lost. As a result the expression of pumps is typically tightly controlled. However, mutants and clinical isolates that over-express efflux pumps are stable and commonly isolated; it may be that such mutants accumulate compensatory mutations allowing them to grow as well as wild-type bacteria.

The molecular basis for the extremely wide substrate specificity of some efflux systems remains an uncertain. It has been suggested that drug substrates share the property of hydrophobic groups in their molecules and that this amphiphilicity was the necessary prerequisite for the substrate to dissolve into the cytoplasmic membrane prior to their transport by the pumps. However, the recent discovery that aminoglycosides, which are polycationic strongly hydrophilic compounds are also effluxed by pumps in P. aeruginosa contradict this hypothesis [3].

Overcoming efflux-mediated resistance

Currently two approaches are being pursued to battle efflux-mediated resistance. To begin with the development of therapeutic agents that inhibit transport activity of efflux pumps, which could be used in combination with existing antibiotics to increase their potency (like β-lactamase inhibitors). Secondly, the modification of existing antibiotics to identify derivatives that are minimally affected by efflux.

Bypassing efflux

A novel class of semi-synthetic tetracyclines, the glycylcyclines [123] exhibit activity against a broad spectrum of Gram-positive and Gram-negative bacteria. Glycylcyclines overcome efflux-mediated resistance because they are not recognized by the transport proteins [123]. The ketolide subclass of macrolides is emerging as an effective alternative to macrolides in treating S. pneumoniae or S. pyogenes. Ketolides even retain activity against strains expressing the MefA/E efflux mechanism, presumably because they are not well exported by this system. Ketolides also appear to be much poorer substrates for the AcrAB-TolC multidrug efflux systems in E. coli than lincosamides and macrolides [29]. Some newly developed fluoroquinolones such as levofloxacin, trovafloxacin, clinafloxacin, moxifloxacin, overcome NorA- or PmrA-mediated efflux in Gram-positive bacteria [108]. They have, however, lost some of their activity against Gram-negative bacteria.

It has not been convincingly shown with any of these agents that the difference in susceptibility is due to their resistance to efflux, rather than to their higher affinity for the target.

Efflux inhibitors

With the increased understanding of the significance of efflux pumps on antibiotic resistance, research is being conducted to discover methods to overcome this mechanism. One of the more practical strategies involves developing compounds that inhibit the efflux mechanism. Inhibition of efflux is potentially one way to improve the clinical efficacy of an antibiotic, even in the presence of target-based mutations, by increasing intracellular antibiotic concentrations. As many efflux pumps posses significant structural homology, it is hoped that one inhibitor compound will be active against a range of pumps from different bacterial species. Several compounds inhibit multidrug pumps of bacteria. For instance, reserpine, a plant alkaloid, is an inhibitor of mammalian efflux pumps as well as Gram-positive pumps such as Bmr and NorA [20;98]. So far, inhibitors of the NorA efflux pump, the Tet-mediated tetracycline efflux have been discovered [138]. As well as broad-spectrum efflux pump inhibitors active against RND pumps in a variety of Gram-negative bacteria, including P. aeruginosa[83;115], E. coli [29], H. influenzae, K. pneumoniae [60] and Campylobacter spp. [86].