Drug Permeation against Efflux by Two Transporters

Abstract

The development of new antibiotics against Gramnegative bacteria is hampered by the powerful protective properties of their cell envelope. This envelope consists of two membranes augmented by efflux transporters, which act in synergy to restrict cellular access to a broad range of chemical compounds. Recently, a kinetic model of this system has been constructed. The model revealed a complex, nonlinear behavior of the system, complete with a bifurcation, and matched very well to experimental uptake data.Here, we expand the model to include multiple transporters and apply it to an experimental analysis of antibiotic accumulation in wild-type and efflux-deficient Escherichia coli. We show that transporters acting across the inner and outer membranes have synergistic effects with each other. In contrast, transporters acting across the same membrane are additive as a rule but can be synergistic under special circumstances owing to a bifurcation controlled by the barrier constant. With respect to ethidium bromide, the inner membrane transporter MdfA was synergistic to the TolC-dependent efflux across the outer membrane. The agreement between the model and drug accumulation was very good across a range of tested drug concentrations and strains. However, antibiotic susceptibilities related only qualitatively to the accumulation of the drugs or predictions of the model and could be fit to the model only if additional assumptions were made about the physiological consequences of prolonged cell exposure to the drugs. Thus, the constructed model correctly predicts transmembrane permeation of various compounds and potentially their intracellular activity.

Inactivation of AdeABC and AdeIJK efflux pumps elicits specific nonoverlapping transcriptional and phenotypic responses in Acinetobacter baumannii

“…Moreover, the MICs of ciprofloxacin, chloramphenicol and cephalosporins remained higher in derivatives of MDR strains, due to the presence of additional resistance mechanisms (Table 1). For the majority of tested antibiotics, the changes in MICs were additive in double knockout strains lacking the activities of both AdeABC and AdeIJK as could be expected for efflux pumps acting across the same membrane (Lee et al ., 2000; Saha et al ., 2020). In contrast, MICs of erythromycin and ciprofloxacin were strain‐specific and were neither multiplicative nor additive but much lower and only in the double knockout strains (Table 1 and Figure 2c).…”

“…The MFS‐type transporter MdfA and AceI from the PACE superfamily are also overproduced in AB5075 adeB ::T26 Δ adeIJK strain (Table 2). In E. coli, MdfA contributes to resistance to fluoroquinolones and chloramphenicol by pumping antibiotics across the inner membrane and acting synergistically with AcrAB‐TolC (Tal and Schuldiner, 2009; Swick et al ., 2011; Saha et al ., 2020). AceI expels polyamines and also some biocides, such as chlorhexidine (Hassan et al ., 2013).…”

Predictive Rules of Efflux Inhibition and Avoidance in Pseudomonas aeruginosa

“…Gram-negative cell envelopes are extremely effective in protecting cells from antibiotics due to the synergy between active efflux pumps and the OM, which creates a permeation barrier with enhanced efficacy (15)(16)(17)(18). The system is characterized by a highly nonlinear behavior leading to efficient efflux of antibiotics that slowly permeate the OM even if they are poor substrates of efflux pumps in biochemical terms.…”

“…MexAB-OprM is the major constitutively expressed pump, which is largely responsible for intrinsic resistance to a variety of antibiotics under laboratory conditions (31)(32)(33). Substrate specificities of these transporters and the efflux constant, K E , which relates to the rates of active and passive efflux of a drug in the range of its low concentrations, are the major drivers of efflux avoidance rules ("efflux rules") (15,16). The unique features of P. aeruginosa and the lack of separation of the efflux and OM contributions in earlier models necessitate the quest for species-specific descriptors and rules of permeation.…”

Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism

“…In extreme cases, bacterial strains have been shown to develop resistance against almost all known chemotherapeutic agents and antibiotics. In Gram negative bacteria, the impermeability of the double membrane is an important determinant of non-specific resistance 1 . The permeability barrier is formed by the combination of the action of protein assemblies, called efflux pumps, that actively export noxious compounds, and of a practically impermeable double membrane barrier composed of lipopolysaccharides, lipids, sugars and peptidoglycans that oppose to the passive diffusion of molecules into the cell 2 .…”