Efflux systems driving resistance and virulence across biological domains

Classification and mechanisms of EPs

Efflux pumps can extrude a wide variety of antimicrobial drugs used against bacteria, fungi, and other pathogens, contributing to reduced efficacy and multidrug resistance. In subsequent sections, we also address their role in extruding antitumor agents. Efflux pumps are membrane transport proteins that extrude compounds from the intracellular environment, contributing significantly to multidrug resistance across a wide range of organisms. These systems are categorized into six major families based on their structure and energy sources (Table 1):

• The ATP-binding cassette (ABC) family uses ATP hydrolysis for active transport.

• The major facilitator superfamily (MFS), small multidrug resistance (SMR) family, and resistance-nodulation-division (RND) family depend on the proton motive force.

• The multidrug and toxic compound extrusion (MATE) family utilizes sodium or proton gradients.

• The most recently identified, proteobacterial antimicrobial compound efflux (PACE) family.

Importantly, ABC transporters represent a converging theme across domains: in bacteria, they extrude a wide variety of antimicrobials, while in cancer cells, they mediate resistance to chemotherapeutics such as taxanes and topoisomerase inhibitors. This dual role emphasizes the translational relevance of efflux inhibition strategies.

Efflux and antimicrobial resistance

EPs contribute to both intrinsic and acquired antimicrobial resistance (AMR) by reducing intracellular drug accumulation, allowing microorganisms to survive in the presence of different compounds. This resistance mechanism is particularly complex due to the remarkable diversity and redundancy among efflux systems. While some EPs, like the MFS transporter TetA, show narrow specificity (e.g., tetracyclines), others, such as the RND system AcrAB-TolC in E. coli are highly promiscuous, expelling fluoroquinolones, β-lactams, aminoglycosides, macrolides, detergents, and dyes (Wright, 2025).

Efflux pumps are widespread among bacteria and can actively extrude structurally diverse antimicrobial agents. This broad substrate range plays a central role in driving multidrug resistance in both Gram-negative and Gram-positive pathogens.

One of the earliest examples was the identification of tetracycline efflux systems in E. coli in the 1980s (McMurry & Levy, 1978; McMurry, Petrucci Jr & Levy, 1980). In Gram-negative bacteria, the combination of EPs and the outer membrane barrier drastically limit drug action. Overexpression of efflux pumps lowers intracellular drug concentrations to subtherapeutic levels, reducing antimicrobial efficacy and promoting treatment failure. This mechanism, first proposed by Levy (1992), was later confirmed across multiple microorganisms (Piddock, 2006; Blair et al., 2015).

In Gram-negative bacteria, efflux pumps belonging to the RND family are major contributors to multidrug resistance. A classic example is the AcrAB-TolC system in Escherichia coli and Salmonella enterica, which exports β-lactams, tetracyclines, fluoroquinolones, and several other antimicrobials air (Blair et al., 2015; Nishino et al., 2021). Other clinically relevant Gram-negative efflux systems include MexAB-OprM in Pseudomonas aeruginosa and AdeABC in Acinetobacter baumannii (Magnet, Courvalin & Lambert, 2001; Coyne, Courvalin & Perichon, 2011). In Gram-positive bacteria, multidrug resistance is largely associated with members of the MFS and ABC families. For instance, the NorA pump in Staphylococcus aureus and PmrA in Streptococcus pneumoniae mediate resistance to fluoroquinolones and macrolides, respectively (Poole, 2001; Kaatz, McAleese & Seo, 2005; Costa et al., 2011). Together, these systems exemplify how efflux pumps in both Gram-negative and Gram-positive pathogens contribute to therapeutic failure.

In mycobacteria, efflux-mediated resistance was first reported in the mid-1990s. Liu, Takiff & Nikaido (1996) identified LfrA, an MFS transporter in Mycobacterium smegmatis, conferring resistance to fluoroquinolones and serving as a model for studies of efflux in M. tuberculosis. This led to the discovery of Tap by Ainsa et al. (1998), which mediates resistance to tetracycline and other agents.

Subsequent studies in M. tuberculosis and M. bovis have identified multiple efflux systems:

• De Rossi et al. (2002) reported 16 putative MFS-encoded transporters

• Silva et al. (2001) characterized P55, implicated in resistance to aminoglycosides, tetracyclines, and macrolides.

• da Silva et al. (2011) highlighted the high number of efflux-related genes in M. tuberculosis, relative to its genome size, emphasizing their relevance in both intrinsic and acquired resistance.

Further studies revealed the importance of RND-type pumps, such as the MmpL family. Notably, MmpL5 is involved in resistance to bedaquiline and clofazimine, while Rv0678 regulates the MmpS5-MmpL5 operon (Andries et al., 2014; Villellas et al., 2017). Interesting, these systems also contribute to bacterial persistence in macrophages (Rodrigues et al., 2013).

Although bacterial efflux has been more extensively characterized, fungal pathogens also deploy highly conserved transporters that significantly contribute to drug resistance, particularly against azole-class antifungals. Candida albicans expresses ABC transporters such as Cdr1 and Cdr2, as well as the MFS-type Mdr1, which collectively contribute to fluconazole resistance and virulence (Coleman & Mylonakis, 2009; Sanguinetti, Posteraro & Lass-Florl, 2015). Similar mechanisms are observed in Aspergillus fumigatus, where transporters such as AtrF and AfuMDR4 are implicated in reduced susceptibility to triazoles (Meneau, Coste & Sanglard, 2016).

These EPs are active in both planktonic cells and biofilms, where they play a critical role in collective protection against antifungal agents. In Candida auris the overexpression of efflux pumps plays a critical role in azole resistance and therapeutic evasion by actively reducing intracellular drug concentrations, thereby compromising treatment efficacy (Zavrel & White, 2015; Rybak et al., 2019).

Efflux and antitumoral resistance

The MDR in cancer cells has been widely investigated since the 1970s, when early studies in Chinese hamster cells revealed cross-resistance to unrelated chemotherapeutic agents such as vinblastine, vincristine, and daunomycin (Biedler & Riehm, 1970; Dano, 1973). These findings pointed to a shared mechanism of resistance, which was later confirmed by the identification of P-glycoprotein (P-gp)—the first ATP-binding cassette (ABC) transporter associated with drug efflux in tumor cells (Juliano & Ling, 1976).

In cancer, EPs of the ABC superfamily are central to treatment failure. The most clinically relevant include:

• P-glycoprotein (P-gp/ABCB1): Overexpressed in multiple cancers such as acute myeloid leukemia, gliomas, hepatocellular carcinoma, and small cell lung cancer, P-gp reduces intracellular concentrations of chemotherapeutic agents, including paclitaxel, doxorubicin, and vincristine. This efflux limits drug efficacy and is associated with poor clinical outcomes (Robey et al., 2018; Zhang et al., 2015).

• Breast cancer resistance protein (BCRP/ABCG2): BCRP extrudes drugs such as mitoxantrone, topotecan, and SN-38. Its overexpression contributes to resistance in breast cancer, acute lymphoblastic leukemia, and gastrointestinal tumors (Nakanishi & Ross, 2012).

• Multidrug resistance-associated proteins (MRPs/ABCC family): MRP1 mediates resistance to anthracyclines, vinca alkaloids, methotrexate, and some tyrosine kinase inhibitors, being frequently overexpressed in lung cancer, neuroblastoma, and pancreatic cancer (Cole, 2014).

Conditions such as hypoxia, low pH, and inflammation can upregulate EP expression or alter drug bioavailability, exacerbating treatment resistance (Robey et al., 2018). Beyond drug extrusion, MDR often results from complex crosstalk between efflux and other resistance-related pathways, including DNA repair mechanisms, evasion of apoptosis, and metabolic reprogramming. For instance, overexpression of ABC transporters such as P-gp and BCRP has been associated with upregulation of DNA repair proteins (e.g., BRCA1/2), altered expression of apoptotic regulators (e.g., Bcl-2, caspases), and increased glycolytic activity that sustains tumor cell survival under therapeutic stress (Gottesman, Fojo & Bates, 2002). This networked resistance phenotype complicates single-agent therapy and underscores the need for combination strategies that simultaneously target efflux activity and complementary pathways (Fletcher et al., 2010; Holohan et al., 2013; Kathawala et al., 2015).

EPs and virulence in fungal pathogens

In addition to their well-established role in antifungal resistance, EPs in fungal pathogens also contribute to virulence by mediating toxin secretion, biofilm formation, and survival within host environments. In Candida albicans, Cdr1 and Cdr2, and Mdr1, have been linked to azole resistance and virulence traits, including increased adhesion, tissue invasion, and biofilm development (Holmes et al., 2008; Prasad & Rawal, 2014). Overexpression of these EPs enhances fungal persistence under host-imposed stress, such as exposure to antimicrobial peptides and oxidative damage. Moreover, in A. fumigatus, MFS-type transporters encoded within secondary metabolite gene clusters are implicated in the export of mycotoxins—compounds that facilitate host damage and immune evasion (Del Sorbo, Schoonbeek & De Waard, 2000; Keller, 2019). These transporters are thought to be activated in response to toxin accumulation, enabling rapid secretion and reducing intracellular toxicity. In both yeasts and filamentous fungi (LaFleur, Kumamoto & Lewis, 2006; Cannon et al., 2009).

Detection efflux by EP inhibitors

Compounds like phenylalanine-arginine β naphthylamide (PAβN) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) are used alongside antimicrobials to evaluate efflux participation. A significant reduction in the minimum inhibitory concentration (MIC) in the presence of an EP inhibitor (EI) is indicative of efflux involvement (Lomovskaya et al., 2001). However, EIs may exert off-target effects and lack specificity, complicating data interpretation (Pages, Amaral & Fanning, 2011).

Fluorometric assays

These assays utilize fluorescent dyes—such as ethidium bromide and Hoechst 33342—that are actively expelled by EPs. Reduced intracellular fluorescence in the presence of these dyes suggests high efflux activity. Fluorometric methods offer real-time, quantitative monitoring and are highly sensitive, making them essential tools for evaluating pump function and inhibitor efficacy (Martins et al., 2011). Confounding variables such as dye toxicity, uptake variability, and non-specific binding can impair result accuracy. Recent innovations, such as more stable fluorescent probes and live-cell imaging techniques, have improved reliability and broadened applicability (Paixao et al., 2009).

Molecular approaches

Molecular approaches are also used for characterizing efflux-mediated resistance. Targeted gene inactivation, using methods such as homologous recombination or CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), allows the functional validation of specific efflux systems by observing changes in susceptibility after gene disruption Additionally, transcriptomic analyses, particularly RNA sequencing enable the assessment of efflux gene expression under various conditions, revealing regulatory responses to antimicrobial exposure. Comparative transcriptomics between wild-type and efflux-deficient strains has been instrumental in identifying induced efflux systems and co-regulated resistance pathways. Together, these strategies provide a comprehensive understanding of efflux mechanisms and support the development of novel therapeutic interventions (Li, Plesiat & Nikaido, 2015; Li et al., 2018).

Microfluidics and single-cell analysis

Microfluidic platforms and single-cell analysis techniques have revolutionized the study of EPs by enabling real-time monitoring of efflux activity at the single-cell level. These methods allow to observe the heterogeneity within bacterial populations, where individual cells may exhibit varying levels of efflux activity due to genetic or environmental factors (Matsumoto et al., 2011). This approach has revealed that even genetically identical cells can display significant differences in efflux activity, which may contribute to the survival of subpopulations under antibiotic pressure (Iino et al., 2012). Single-cell analysis also provides insights into the dynamics of EP regulation and the role of stochastic gene expression in resistance development (Blair & Piddock, 2016).

Biosensors

Genetically engineered biosensors are being developed to detect efflux activity with high specificity and sensitivity. These biosensors typically consist of reporter genes, such as fluorescent or luminescent markers, that are activated in response to EP function. A small molecule-responsive biosensor was developed to link PDR gene activation to growth in Saccharomyces cerevisiae. Using diverse inducers and a homozygous diploid deletion library, genome-wide screens identified both known and novel regulators of PDR. Notably, genes involved in the mitotic spindle assembly checkpoint were found to modulate EP expression, revealing a new connection between PDR and cell cycle control. Biosensors offer several advantages, including rapid detection, high throughput, and the ability to monitor efflux activity in live cells without disrupting their physiology (Li et al., 2019).

Advanced imaging techniques

Techniques such as super-resolution microscopy and cryo-electron microscopy (cryo-EM) are providing unprecedented insights into the structure and function of EPs. For example, cryo-EM has been used to visualize the three-dimensional structure of the AcrAB-TolC complex in E. coli, revealing how the pump undergoes conformational changes to transport substrates across the bacterial membrane (Du et al., 2018; Klenotic, Morgan & Yu, 2021). These structural insights are critical for the rational design of EIs that can block EP function without affecting essential cellular processes.

In summary, emerging technologies are transforming the study of efflux-mediated resistance, offering new opportunities to understand the mechanisms of efflux develop targeted therapies, and combat the global threat of multidrug-resistant infections. However, their successful implementation will require interdisciplinary collaboration, investment in infrastructure, and the integration of experimental and computational approaches.

Mechanisms and potential candidates

A variety of strategies have been proposed to inhibit drug efflux and restore antimicrobial efficacy. These include direct inhibition of EPs by competitive or non-competitive EIs, disruption of EP assembly, and interference with the proton motive force that drives secondary active transporters. Other approaches focus on the transcriptional regulation of efflux genes, aiming to suppress pump expression. Additionally, structural modification of antimicrobial agents to evade efflux recognition, or trapping transporters in inactive conformations, represents promising complementary tactics. EP inhibitors, especially when used as adjuvants, offer a powerful means to restore the effectiveness of existing antimicrobials and delay the emergence of resistance, reinforcing the therapeutic value of targeting efflux as part of a comprehensive antimicrobial strategy. The mechanism more frequently described is competitive inhibition, where EIs bind directly to the substrate-binding site of the EP, preventing the actual substrate from accessing and being extruded. This direct competition effectively reduces the efflux of antimicrobial agents, increasing their intracellular concentrations, and if used as an adjuvant with an antimicrobial, will reduce its effective dose. For instance, certain inhibitors have been shown to occupy the substrate-binding pocket of the AcrB EP in E. coli, thereby blocking drug efflux (Kim et al., 2021b).

Disruption of EP assembly was showed through EIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antimicrobials in E. coli and other Gram-negative bacteria (Abdali et al., 2017).

Another strategy involves allosteric modulation, wherein EIs bind to sites distinct from the substrate-binding region, inducing conformational changes that impair the pump’s function. This alteration can decrease the pump’s affinity for its substrates or disrupt the conformational changes necessary for substrate transport. Recent studies have identified pyridylpiperazine-based compounds that act as allosteric inhibitors of RND-type multidrug EPs, effectively hindering their activity (Zgurskaya et al., 2021).

Energy disruption represents another approach, targeting the energy sources essential for pump operation. EPs often rely on the proton motive force or ATP hydrolysis to actively transport substrates across the membrane, EIs can effectively inhibit efflux activity (Sharma, Gupta & Pathania, 2019; Li et al., 2024).

It was identified that both synthetic and natural EIs with improved efficacy and specificity have been reported, mainly in microbial models. However, EIs are also being actively investigated in oncology, with several candidates—such as tariquidar, Ko143, and SCO-101—under evaluation for their ability to reverse multidrug resistance in cancer cells, including in ongoing clinical trials. For instance, MBX-2319, a synthetic pyranopyridine derivative, targets the AcrAB-TolC system in E. coli, enhancing the bactericidal activity of ciprofloxacin (Opperman et al., 2014). Similarly, natural compounds such as baicalein have been found to inhibit EPs in S. aureus, restoring the effectiveness of antimicrobial agents (Mao et al., 2023).

An alternative strategy to inhibit EP activity involves repressing the expression of corresponding genes. This approach employs antisense oligonucleotides or small interfering RNA (siRNA) to selectively prevent the transcription or translation of genes encoding EPs (Kong et al., 2015). Further, some efflux inhibitors, including tariquidar and verapamil, have been studied in both microbial and oncological contexts, highlighting the overlap between antimicrobial resistance and cancer multidrug resistance. This convergence suggests that lessons learned in one field may inform the other, with microbial models providing mechanistic insights and oncology trials offering translational evidence for safety and efficacy. Such cross-domain perspectives reinforce the potential of efflux inhibition as a unifying therapeutic strategy.

CRISPR/Cas9-Mediated EP modulation: a novel strategy against multidrug resistance

The CRISPR/Cas9 system is a gene-editing technology derived from a bacterial adaptive immune mechanism. Originally used by bacteria and archaea to defend against invading genetic elements, this system has been adapted as a powerful and precise tool for genome editing in eukaryotic and prokaryotic cells (Vaghari-Tabari et al., 2022). CRISPR/Cas9 enables targeted modification, disruption, or regulation of specific genes, and has been increasingly explored as a strategy to combat antimicrobial resistance by interfering with EP expression and function.

Collectively, these diverse strategies underscore the potential of EIs to combat MDR by targeting efflux mechanisms through competitive inhibition, allosteric modulation, energy disruption, and gene expression. Recent studies have demonstrated that CRISPR/Cas9-mediated disruption of these efflux transporter genes can restore antimicrobial and antitumoral activity (Vaghari-Tabari et al., 2022; Sajid, Rahman & Ambudkar, 2023).

In bacteria, CRISPR interference (CRISPRi)—a variant of CRISPR/Cas9 that represses transcription without DNA cleavage—has been used to silence EP genes. Wan et al. (2022) developed a CRISPRi system targeting the AcrAB-TolC pump in E. coli, resulting in substantial downregulation of efflux gene expression and increased susceptibility to multiple antibiotics, including rifampicin, erythromycin, and tetracycline. Additionally, this system reduced biofilm formation, further enhancing antimicrobial efficacy.

More broadly, CRISPR/Cas systems have been included among non-traditional strategies to overcome antimicrobial resistance. Their integration into combinatorial therapies, together with antimicrobial peptides, phage therapy, and EP inhibitors has opened new avenues for overcoming resistance in both clinical and experimental settings (Konwar et al., 2022).

Natural products as EIs candidates

Natural products have emerged as a promising source of EIs due to their structural diversity and potential for synergistic effects with existing antimicrobials. For example, 5′-methoxyhydnocarpin-D is a flavonolignan produced by species like Berberis fremontii, Berberis repens, and Berberis aquifolia that can enhance the antibacterial activity of ciprofloxacin and tobramycin against S. aureus (Gaurav et al., 2023). Similarly, curcumin, a polyphenol from turmeric (Curcuma longa), has demonstrated to inhibit EPs in P. aeruginosa, reducing resistance to ciprofloxacin (Negi et al., 2014).

Baicalein is a flavonoid from Scutellaria baicalensis was recently described inhibitor EPs in S. aureus, reducing resistance to fluoroquinolones (Moulick & Roy, 2024). Carvacrol and Thymol are phenolics compounds found in Origanum vulgare and Thymus vulgaris inhibits NorA EP in MDR S. aureus strains (Dos Santos Barbosa et al., 2021).

Reserpine is an alkaloid obtained from Rauvolfia serpentina described almost 30 years ago, inhibits EPs in Gram-positive bacteria, including S. aureus, S. pneumoniae and M. tuberculosis (Angelini, 2024; Schmitz et al., 1998; Garvey et al., 2011; Huang et al., 2013). Plumbagin is a naturally occurring napthoquinone, obtained from Plumbago zeylanica is known to modulate cellular proliferation, carcinogenesis, and radio-resistance and is also a cardiotonic, hepatoprotective, and neuroprotective agent. Also, has been shown to inhibit ABCG2 EP that is one of the reasons for the development of MDR in cancer (Shukla et al., 2007).

Microbial metabolites also have emerged as a promising source of EIs, Ethyl 4-bromopyrrole-2-carboxylate from the soil bacterium Streptomyces sp. demonstrates efficacy of the antibiotic-EI combination against P. aeruginosa. An indole (RP2) isolated from the soil bacterium has showed potential of EI in resistance in S. aureus (Tambat et al., 2019; Tambat et al., 2022).

Synthetic compounds as EIs

Several synthetic compounds have been evaluated as EIs, with varying degrees of success. For example, MC-207,110 (also known as PaβN: Phe-Arg β-naphthylamide) is one of the earliest synthetic EIs studied. It has shown efficacy in inhibiting RND-type EPs in P. aeruginosa and E. coli, reversing resistance to fluoroquinolones and β-lactams (Lomovskaya et al., 2001). However, its clinical use is limited due to toxicity and off-target effects. Another notable synthetic EI is MBX2319, a pyridopyrimidine derivative that specifically targets the AcrAB-TolC efflux system in E. coli and other Enterobacteriaceae bacteria. MBX2319 has shown promise in preclinical studies, significantly reducing the MIC of antibiotics like ciprofloxacin and levofloxacin (Opperman et al., 2014). However, it has not yet advanced to clinical trials.

D13-9001 is an EI that targets the MexAB-OprM efflux system in P. aeruginosa which enhances the activity of β-lactams and fluoroquinolones. This compound has demonstrated potent efflux inhibitory activity in vitro and animal models, but its clinical development has been hindered by challenges related to pharmacokinetics and toxicity (Nakashima et al., 2013). A novel EI MBX-4191that targets the AcrAB-TolC system in E. coli, restored susceptibility to multiple antimicrobials (Vargiu et al., 2014). Tetrahydropyridines can act as a potential pharmacophore candidate with potential in vitro and in silico as inhibitor of efflux in E. coli and M. abscessus (Silva Jr et al., 2017; Vianna et al., 2019).

Pyrvinium, an anthelmintic traditionally used to treat pinworm infections, has shown promising antimicrobial properties. It was found to be effective against preformed biofilms and demonstrated excellent in vivo efficacy. When combined with ciprofloxacin, pyrvinium significantly enhanced activity against multidrug-resistant S. aureus, even at clinically achievable concentrations. This synergistic interaction suggests that the pyrvinium–ciprofloxacin combination could be a valuable strategy for treating persistent and biofilm-associated infections caused by MDR pathogens (Mahey et al., 2021). Nilotinib, a tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia, has also emerged as a potential EP inhibitor, was shown to specifically inhibit the NorA EP in S. aureus when used in combination with ciprofloxacin. This inhibition significantly enhanced the antimicrobial activity of ciprofloxacin, particularly in the context of biofilms (Zimmermann et al., 2019).

A summary of representative natural and synthetic efflux inhibitors, their main targets, and contexts of use is provided in Table S1.