Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications

Abstract

Pseudomonas aeruginosa is a notorious multidrug‐resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug‐resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.

This work comprehensively summarized the regulatory networks of the major porin proteins and efflux pumps that play critical roles in antibiotic influx and efflux in Pseudomonas aeruginosa. It also discussed promising therapeutic approaches to combat multidrug‐resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps.

INTRODUCTION

Antibiotic resistance is a serious and growing threat to the global public health. It has been estimated that as many as 10 million people could die annually from antibiotic‐resistant infections by 2050 if proactive solutions are not implemented to slow down the current rising trend of antibiotic resistance (Jim, 2016). Nosocomial ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, are designated as “priority status” in the World Health Organization's list of pathogens for which the development of new antibiotics is urgently needed (De Oliveira David et al., 2020). Widely distributed in soil and aquatic environments, P. aeruginosa is an opportunistic pathogen that is responsible for 10–15% of nosocomial infections worldwide and often causes severe even life‐threatening infections in immunocompromised individuals (Crone et al., 2020; Shi et al., 2019). In particular, P. aeruginosa is the predominant pathogen causing lung infections in people with cystic fibrosis (PwCF), and chronic infections caused by antibiotic‐resistant P. aeruginosa frequently result in declined pulmonary functions as well as high mortality in these patients (Sousa & Pereira, 2014). Infections caused by P. aeruginosa are one of the top threats to antibiotic resistance worldwide, which lead to more than 300,000 deaths in a year associated with antibiotic resistance (Murray et al., 2022; Sastre‐Femenia et al., 2023).

MECHANISMS OF ANTIBIOTIC RESISTANCE IN P. aeruginosa

In general, antibiotics must penetrate the bacterial cell membrane and then exert their activity by interacting with specific cellular targets. For example, aminoglycoside antibiotics such as gentamicin and amikacin interact with ribosomal 30S subunits to inhibit protein translation, fluoroquinolone antibiotics such as levofloxacin and ciprofloxacin interact with DNA gyrase and topoisomerase IV to inhibit DNA replication, β‐lactam antibiotics such as carbapenems interact with enzymes involved in peptidoglycan synthesis to inhibit cell wall synthesis, and polymyxin antibiotics such as colistin interact with lipopolysaccharides (LPS) to increase membrane permeability and also induce hydroxyl‐mediated cell toxicity (Halawa et al., 2024). Accordingly, bacteria including P. aeruginosa have evolved various mechanisms of resistance to evade the inhibitory or killing effects of antibiotics by preventing the entry of antibiotics, actively extruding antibiotics outside the cell, degrading or modifying antibiotic molecules, and protecting or modifying cellular targets (Darby et al., 2023).

These mechanisms of resistance can be either intrinsic, acquired, or adaptive (Pang et al., 2019). Intrinsic resistance is an inherent action that enables the bacterial cell to mitigate the threat of antibiotics. For example, P. aeruginosa produces antibiotic‐inactivating enzymes such as β‐lactamases that degrade β‐lactam antibiotics (Glen & Lamont, 2021). In addition, the failure of antibiotic‐based treatment of P. aeruginosa infections is largely dependent on two important intrinsic resistance determinants in this pathogen. One of them is the restricted membrane permeability due to the deficiency of porins for non‐specific substrates and the other one is the presence of abundant efflux pumps that actively export a wide range of antibiotics with high dissimilarities in structures (Chevalier et al., 2017; Li & Plésiat, 2016). Acquired resistance is generated by the acquisition of exogenous genetic elements that contain resistance genes (Munita Jose & Arias Cesar, 2016). Since antibiotic resistance genes can be carried by mobile genetic elements such as plasmids, transposons, and phages, it is easy for bacteria to acquire antibiotic‐resistant genes through horizontal gene transfer. For example, the acquisition of genes encoding β‐lactamases, which confer resistance to β‐lactam antibiotics, has been frequently reported in P. aeruginosa (Kabic et al., 2023). Acquired resistance is also achieved through mutational changes in resistance genes. Mutations in the repressors of efflux pumps are ubiquitous in clinical P. aeruginosa isolates, leading to the overexpression of efflux pumps and the induced resistance to multiple antibiotics (Esquisabel et al., 2011). Adaptive resistance is a transient elevation of resistance when bacterial cells encounter specific environmental signals or their growth conditions are changed (Coleman Shannon et al., 2020). Adaptive resistance is commonly the result of the changed expression levels of intrinsic resistance determinants, such as suppression of the antibiotic influx machinery or induction of the antibiotic efflux machinery under special growth conditions, which endows the pathogen an elevated ability to survive better with increasing concentrations of antibiotics (Fernández & Hancock Robert, 2012). In addition, adaptive resistance in P. aeruginosa is also linked to the formation of biofilms which are structurally complicated communities of bacteria that form an extracellular barrier to protect the cells inside and enable them to evade the killing effect of antibiotics (Ciofu & Tolker‐Nielsen, 2019).

Pseudomonas aeruginosa harbours a relatively large genome, which exhibits great plasticity and adaptability (Klockgether et al., 2011; Kung Vanderlene et al., 2010). The genome encodes a very large number of regulatory components such as transcription factors and two‐component systems (TCSs) (Shao et al., 2023; Stover et al., 2000). These genetic features allow the pathogen to quickly arm with sufficient antibiotic resistance determinants in response to various innate or environmental signals and then persist during antibiotic‐based treatments. Understanding the regulatory mechanisms of the inducible resistance will provide valuable therapeutic implications. Hence, regulation of the resistance in P. aeruginosa has been a subject of intensive investigations in the past two decades. Considering the critical contributions of both the restricted antibiotic influx and active antibiotic efflux to the multidrug resistance of P. aeruginosa, in this review, we will next focus on the summary of the current research advancements in the regulation of antibiotic influx porins and efflux pumps.

PORINS IN P. aeruginosa

The relatively impermeable double‐membrane structure of Gram‐negative bacteria compared to Gram‐positive bacteria makes it difficult for a number of antibiotics to penetrate into the cell (Masi et al., 2017). Porins are β‐barrel protein channels located in the outer membrane of Gram‐negative bacteria, and most of them allow the entrance of hydrophilic compounds into cells (Henderson et al., 2016). These channels play a critical role in the uptake of nutrients and also provide the opportunity for antibiotic influx, although some nutrients and antibiotics can also enter cells directly through the outer membrane lipid bilayer (Rollauer et al., 2015; Ude et al., 2021). For example, β‐lactam antibiotics enter bacterial cells mainly through porins (Prajapati et al., 2021). In the past decades, important efforts have been made to unveil the structure and function of porins in P. aeruginosa (Chevalier et al., 2017). It is noticed that porins in P. aeruginosa allow smaller molecules which are generally <200 Da to pass through, and the membrane permeability of P. aeruginosa is about 100‐fold lower than that of another Gram‐negative pathogen Escherichia coli (Hancock & Brinkman, 2002; Yoshimura & Nikaido, 1982).

Multiple porins have been identified and characterized in P. aeruginosa. OprF, the most abundant non‐lipoprotein outer membrane protein, plays an important role in maintaining the integrity of the outer membrane and is involved in many physiological processes such as biofilm formation and pathogenesis (Moussouni et al., 2021). It is a homologue of the outer membrane protein A (OmpA) in E. coli and is known as a non‐specific aqueous channel for the main uptake of ions and saccharides but shows low efficiency for antibiotics (Reusch, 2012). Besides OprF, the remaining known porins in P. aeruginosa are substrate specific, including two small outer membrane proteins OprG and OprH which are, respectively, responsible for the transportation of small amino acids and divalent cations (Bell et al., 1991; ReddySanganna Gari et al., 2018), two OprB glucose porins, OprB and OprB2 (Wylie & Worobec, 1995), the phosphate porin OprP (Sukhan & Hancock, 1995), the pyrophosphate porin OprO (Siehnel et al., 1992), and the OprD‐family porins which consist of 8 OccD members and 11 OccK members (Tamber et al., 2006). Among the substrate‐specific porins, OprD (OccD1) is the most well‐investigated one since it not only translocates basic amino acids and peptides but also supports the entry of carbapenem antibiotics, especially imipenem and meropenem, into the cell, serving as an important target for antibiotic therapy (Li et al., 2012; Wolter et al., 2004). Next, the regulation of OprD and another porin OprH which contributes to antibiotic influx indirectly by interaction with LPS was summarized and introduced.

Regulation of OprD

A number of studies have shown that inactivation of OprD due to mutations inside the open reading frame (ORF) and reduced expression of OprD due to mutations in the promoter or its regulators are frequently observed in clinical P. aeruginosa isolates (Biggel et al., 2023; Sherrard et al., 2022; Shu et al., 2017). Inactivation of OprD contributes significantly to the carbapenem resistance of P. aeruginosa. In addition to the mutations, the expression of OprD is reversibly controlled by multiple regulators in response to diverse environmental signals (Figure 1).

FIGURE 1.

The regulatory network of the porin protein OprD in Pseudomonas aeruginosa. The expression of oprD is regulated by ArgR (signal: arginine (Arg)), FliA, SigX, RpoN, MexT, CzcS/CzcR (signal: Zn²⁺), CopS/CopR (signal: Cu²⁺), ParS/ParR, Hfq, and histidine (His), glutamate (Glu), alanine (Ala) at the transcriptional level, and ErsA, Sr0106, Hfq at the post‐translational level. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter or mRNA; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; IM, inner membrane; OM, outer membrane.

The physiological function of OprD was first identified as a transporter for basic amino acids, and the expression of oprD was found to be induced by arginine, histidine, glutamate, or alanine (Ochs Martina, Lu, et al., 1999). The arginine‐induced expression of oprD was abolished when the gene encoding the arginine‐responsive AraC/XylS‐family regulator ArgR was deleted (Ochs Martina, Lu, et al., 1999). Further gel mobility shift and DNase assays demonstrated that ArgR induced the expression of oprD through the direct binding of ArgR to its promoter (Ochs Martina, Lu, et al., 1999). In addition to its physiological substrates, the expression of oprD is modulated by metal ions. For instance, exposure of P. aeruginosa to Zn²⁺ or Cu²⁺ exerted negative effects on the expression of oprD, resulting in increased levels of resistance to carbapenem antibiotics (Caille et al., 2007; Perron et al., 2004). CzcS/CzcR, a TCS activated by Zn²⁺ to confer P. aeruginosa tolerance to divalent metals such as Zn²⁺, Cd²⁺, and Co²⁺, was found to modulate the expression of oprD through the direct binding of CzcR to its promoter (Dieppois et al., 2012; Perron et al., 2004). Although CzcR can directly bind to the promoter of oprD to inhibit its transcription, an RNA chaperone Hfq was found to be necessary for the localization of CzcR to the promoter of oprD (Ducret et al., 2016). Like CzcS/CzcR, another TCS CopS/CopR which is induced by Cu²⁺ and involved in Cu²⁺ tolerance was shown to negatively regulate the expression of oprD, preventing the uptake of carbapenem antibiotics and causing P. aeruginosa resistance to carbapenem antibiotics (Caille et al., 2007). Similarly, CopS/CopR also requires Hfq to control oprD expression (Ducret et al., 2016). In addition, expression of oprD is repressed by another TCS named ParS/ParR (Wang et al., 2013).

MexT is a LysR‐type regulator that plays an important role in activating the efflux pump MexEF‐OprN. When it was overproduced, it not only caused an increased expression level of the mexEF‐oprN efflux operon but also inhibited the expression of oprD (Ochs Martina, McCusker Matthew, et al., 1999). Sigma factors are critical for transcription initiation, which confers the specificity of promoter recognition by RNA polymerase. ChIP‐Seq assays have identified that sigma factors such as SigX, RpoN, and FliA directly interact with the promoter of oprD and play an important role in activating the expression of oprD (Schulz et al., 2015; Shao et al., 2018). Expression of oprD is also controlled at the post‐transcriptional level by Hfq, which functions together with two sRNAs (Sr0161 and ErsA) to base pair with the 5′ untranslated region (UTR) of oprD, leading to the reduced production of OprD and increased resistance of P. aeruginosa to meropenem (Sonnleitner et al., 2020; Zhang et al., 2017).

Regulation of OprH

OprH, the smallest porin in P. aeruginosa responsible for the uptake of divalent cations, interacts with LPS through electrostatic interactions and then tightens the outer membrane (Edrington et al., 2011; Kucharska et al., 2016). Unlike β‐lactam antibiotics, which cross bacterial membranes through porins, polymyxin and aminoglycoside antibiotics penetrate the cell membrane by interacting with the negatively charged LPS (Saxena et al., 2023). Therefore, overexpression of OprH prevents the binding of these antibiotics to LPS and the entry of them into the cell, conferring P. aeruginosa resistance to polymyxin and aminoglycoside antibiotics (Saxena et al., 2023).

OprH is genetically linked to the TCS PhoP/PhoQ that regulates the expression of the oprH‐phoP‐phoQ operon (Figure 2) (Macfarlane et al., 1999). Since the activity of PhoP/PhoQ is induced during Mg²⁺ starvation, increased resistance to polymyxin and aminoglycoside antibiotics owing to the overproduction of OprH was observed during Mg²⁺ starvation (Macfarlane et al., 1999, 2000). In contrast, high concentrations of Ca²⁺ led to reduced expression levels of oprH‐phoP‐phoQ and consequently increased sensitivity to polymyxin and aminoglycoside antibiotics (Guragain et al., 2016). The TCS BqsS/BqsR (CarS/CarR) regulates Ca²⁺ homeostasis and connects the expression of oprH‐phoP‐phoQ to the Ca²⁺ signal (Guragain et al., 2016). Interestingly, BqsS/BqsR responds not only to the Ca²⁺ signal but also to the Fe²⁺ signal. When the concentration of Fe²⁺ was elevated, the activity of BqsS/BqsR was induced and the expression of oprH was upregulated subsequently (Kreamer Naomi et al., 2015). Moreover, proteomic analysis showed that the production of OprH in P. aeruginosa was positively controlled by the TCS PirS/PirR which senses plant‐derived phenols with Fe‐complexing capacities (Luscher et al., 2022).

FIGURE 2.

The regulatory network of the porin protein OprH in Pseudomonas aeruginosa. The expression of oprH is regulated by BqsS/BqsR (signals: Ca²⁺ and Fe²⁺), PhoQ/PhoP (signal: Mg²⁺), BrlR, p‐anisaldehyde, agmatine, putrescine, and contacts with bronchial epithelial and lung epithelial cells at the transcriptional level. Production of OprH induced by citrate and PirS/PirR was revealed in proteomic studies. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; IM, inner membrane; OM, outer membrane.

In addition to TCSs that sense metal or metal‐related signals, the MerR‐like regulator BrlR was demonstrated to inhibit the expression of oprH and increase the sensitivity of P. aeruginosa to colistin by directly binding to the promoter of the oprH‐phoP‐phoQ operon (Chambers Jacob & Sauer, 2013). Citrate was found to increase the abundance of OprH in a quantitative proteomic study in which P. aeruginosa was exposed to different carbon sources during growth (Sauvage et al., 2022). Expression of oprH is also modulated by polyamines and the contact of P. aeruginosa with bronchial or lung epithelial cells. For example, putrescine and agmatine were reported to greatly induce the expression of oprH and the resistance of P. aeruginosa to polymyxin and aminoglycoside antibiotics (Kwon Dong & Lu, 2006). The contact of P. aeruginosa with human bronchial epithelial cells substantially increased the expression of the oprH‐phoP‐phoQ operon, while intriguingly, it was also reported that the contact of P. aeruginosa with the lung epithelial cells led to a reduced expression of oprH (Chugani & Greenberg, 2007; Gellatly Shaan et al., 2012). In addition, the expression of oprH is induced by a plant‐derived compound p‐anisaldehyde (Adewunmi et al., 2020).

EFFLUX PUMPS IN P. aeruginosa

In addition to changing the abundance of porin proteins, the presence of a broad variety of efflux pumps further reduces the intracellular content of antibiotics in P. aeruginosa, exacerbating the level of antibiotic resistance. Active extrusion by efflux pumps constitutes an important factor contributing to the resistance of P. aeruginosa to antibiotics from different categories (Lorusso et al., 2022). Efflux pumps associated with antibiotic resistance commonly belong to the RND (resistance–nodulation–division), MFS (major facilitator superfamily), MATE (multidrug and toxic compound extrusion), SMR (small multidrug resistance), and ABC (ATP‐binding cassette) superfamilies or families (Sun et al., 2014).

Efflux pumps from the RND family, which are major exporters contributing to the intrinsic antibiotic resistance, are located in the inner membrane (IM) and interact with the periplasmic adaptor protein (also called membrane fusion protein) and the outer membrane (OM) channel, producing a tripartite complex spanning the IM, the periplasm, and the OM (Puzari & Chetia, 2017). In P. aeruginosa, the RND‐family efflux pumps play a critical role in extruding antibiotics from both the periplasm and the cytosol to the environment. Generally, the cytoplasmic and periplasmic components of the RND efflux pumps in P. aeruginosa are named Mex and the component in the OM is named Opr (Pang et al., 2019). Twelve RND efflux pumps have been identified in P. aeruginosa. Among them, four efflux pumps, MexAB‐OprM, MexCD‐OprJ, MexEF‐OprN, and MexXY‐OprM (OprA), contribute significantly to the resistance of P. aeruginosa to multiple antibiotics, and the regulatory mechanisms of these four efflux pumps are summarized and introduced in this section (Table 1).

TABLE 1.

A summary of regulators and signals that modulate the expression efflux pump genes in Pseudomonas aeruginosa.

Name	Regulator	Signals or stimuli	References
MexAB‐OprM	MexR	Oxidative stress, sulfane sulfur	(Chen et al., 2008; Xuan et al., 2020)
	CpxS/CpxR	Envelope stress	(Tian & Wang, 2023)
	CzcS/CzcR	Zn2+	(Chen et al., 2023)
	NalD	?	(Morita, Cao, et al., 2006)
	BrlR	?	(Liao et al., 2013)
	PA3225	?	(Hall Clayton et al., 2017)
	NalC	Cinnamaldehyde, chlorinated phenols	(Ghosh et al., 2011; Tetard et al., 2019)
	AmpR	?	(Balasubramanian et al., 2012)
	VqsM	?	(Dong et al., 2005)
	ArmR	?	(Wilke et al., 2008)
	RosS1/RosS2/RosA2	?	(Sivaneson et al., 2011)
	?	C4‐HSL	(Sawada et al., 2004)
	?	Ca2+	(Khanam et al., 2017)
	?	Piperine	(Liu et al., 2023)
MexCD‐OprJ	NfxB	?	(Shiba et al., 1995)
	EsrC	?	(Purssell et al., 2015)
	AlgU	Envelope stress	(Fraud et al., 2008)
	spae3959.1, spae3706.1, spae1558.1	Doxycycline	(Zhang et al., 2022)
	VqsM	?	(Liang et al., 2014)
	?	LL‐37	(Strempel et al., 2013)
	?	Phenylethylamine	(Muñoz‐Cazalla et al., 2023)
	?	Tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G, acriflavine	(Morita et al., 2001)
	?	Benzalkonium chloride, chlorhexidine gluconate	(Morita et al., 2003)
	?	Dequalinium chloride, procaine	(Laborda et al., 2019)
MexEF‐OprN	MexT	?	(Maseda et al., 2000)
	BrlR	?	(Liao et al., 2013)
	RpoA	?	(Cai, Lu, et al., 2023)
	GcsR	?	(Sarwar et al., 2016)
	CmrA	Electrophilic stress	(Juarez et al., 2017)
	PA2048	?	(Juarez et al., 2017)
	MexS	?	(Fargier et al., 2012)
	MxtR	?	(Zaoui et al., 2012)
	ParS/ParR	?	(Wang et al., 2013)
	AmpR	?	(Balasubramanian et al., 2012)
	MvaT	?	(Westfall et al., 2006)
	?	Paerucumarin	(Iftikhar et al., 2020)
	?	Cinnamaldehyde	(Tetard et al., 2019)
	?	Z‐ethylthio enynone	(Kristensen et al., 2024)
MexXY	MexZ	?	(Matsuo et al., 2004)
	PmrA/PmrB	Halogenated indoles	(Dou et al., 2023)
	ParS/ParR	Zn2+	(Poole et al., 2019)
	AmgS/AmgR	Envelope stress	(Krahn et al., 2012)
	ArmZ	?	(Kawalek et al., 2019)
	PNPase	?	(Fan et al., 2021)
	RplY	?	(El'Garch et al., 2007)
	Fmt, FolD	?	(Caughlan Ruth et al., 2009)
	RpmA, RplU	?	(Lau Calvin et al., 2012)
	SuhB	?	(Shi et al., 2015)
	PA2798	?	(Genova et al., 2023)
	HtpX, PA5528	?	(Lau et al., 2015)
	?	Norspermidine	(Bolard et al., 2019)
	?	Oxidative stress	(Fraud & Poole, 2011)

Note: Regulator names in bold indicate the direct binding of these regulators to the promoters of efflux operons.

Regulation of MexAB‐OprM

MexAB‐OprM, the first reported antibiotic efflux pump in P. aeruginosa, is responsible for the extrusion of a broad range of antibiotics such as β‐lactams, fluoroquinolones, macrolides, chloramphenicol, etc. (Poole et al., 1993). Normally, MexAB‐OprM is constitutively active in the wild‐type P. aeruginosa strains and upregulation of MexAB‐OprM substantially contributes to the development of P. aeruginosa resistance to multiple antibiotics (Sobel Mara, Hocquet, et al., 2005). Notably, genes or operons encoding efflux pumps are frequently linked to a regulatory gene whose product serves as a local regulator controlling the expression of the efflux genes. MexR is encoded upstream of the mexAB‐oprM operon and acts as the local transcriptional repressor that negatively regulates the mexAB‐oprM operon as well as its own expression (Figure 3) (Poole, Tetro, et al., 1996). Mutations in mexR and premature termination of the MexR peptide are frequently found in clinical P. aeruginosa strains, which is the major cause of the de‐repression of mexAB‐oprM and the multidrug resistance of P. aeruginosa (Xu et al., 2019). For instance, the nalB‐type isolates of P. aeruginosa displaying an increased level of resistance to β‐lactams are found to exhibit an elevated expression level of the MexAB‐OprM efflux pump due to mutations in the mexR gene (Saito et al., 1999).

FIGURE 3.

The regulatory network of the efflux pump MexAB‐OprM in Pseudomonas aeruginosa. MexR is a local repressor of MexAB‐OprM. The expression of mexAB‐oprM is regulated directly or indirectly by NalC (signals: cinnamaldehyde, chlorinated phenols), ArmR, VqsM, AmpR, RocS1/RocS2/RocA2, PA3225, NalD, BrlR, CxpS/CxpR (signal: envelope stress), CzcS/CzcR (signal: Zn²⁺), Ca²⁺, C4‐HSL, sulfane sulfur, piperine, and oxidative stress at the transcriptional level. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; IM inner membrane; OM, outer membrane.

Regulators or signal molecules can also regulate the expression of mexAB‐oprM indirectly by modulating the expression of the local regulator MexR. AmpR, a LysR‐family regulator controlling the expression of the chromosomal β‐lactamase AmpC, inhibits the expression of MexR by possibly binding to the promoter region of the mexR gene, which causes the overproduction of the MexAB‐OprM efflux pump and the increase of resistance to antibiotics (Balasubramanian et al., 2012). RNA‐seq analysis showed that loss of the AraC‐type transcriptional regulator VqsM led to 2.9‐fold upregulation of the mexR gene, but how VqsM repressed the mexR gene remains unclear (Dong et al., 2005). Some extracts from traditional Chinese herbs display promising activity in inhibiting the expression of the mexAB‐oprM efflux operon through the upregulation of mexR. For example, piperine is a monomer extracted from the Chinese herb pepper, and it was found that PIP could significantly promote the production of MexR to inhibit the production of the MexAB‐OprM efflux pump and accumulate imipenem in the cell (Liu et al., 2023).

In addition to the changed transcription levels of MexR, the activity of MexR is influenced by the anti‐repressor ArmR and multiple environmental stresses and chemical signals. ArmR is an anti‐repressor which can interact with MexR physically to form an ArmR–MexR complex, which sequesters the MexR protein from binding to the promoter of the mexAB‐oprM operon (Wilke et al., 2008). NalC is a TetR‐family repressor that negatively regulates the expression of ArmR by directly binding to the promoter region of PA3720‐armR (Jeong et al., 2023). Therefore, when NalC is repressed or inactivated, the expression of mexAB‐oprM can be induced indirectly through the overproduced ArmR (Cao et al., 2004). For example, the main constituents of cinnamon bark oil cinnamaldehyde and chlorinated phenols were demonstrated to induce the expression of mexAB‐oprM as well as multidrug resistance of P. aeruginosa by repressing the expression of nalC and inactivating the promoter‐binding ability of NalC, respectively (Ghosh et al., 2011; Tetard et al., 2019). Owing that two Cys residues (Cys‐30 and Cys‐62) in MexR are redox‐active, oxidative stress induces the formation of intermonomer disulfide bonds involving Cys30 and Cys62, causing the dissociation of MexR from the target promoter of the mexAB‐oprM operon and the overproduction of the MexAB‐OprM efflux pump (Chen et al., 2008). P. aeruginosa can produce cellular sulfane sulfur from L‐cysteine metabolism or H₂S oxidation by sulfide: quinone oxidoreductase (Li et al., 2019). Sulfane sulfur was determined as an intrinsic signal that directly reacts with MexR, which causes the dissociation of MexR from the target promoter through forming disulfide (‐SS‐) or trisulfide (‐SSS‐) cross‐links between the two Cys residues and then de‐represses the mexAB‐oprM operon (Xuan et al., 2020).

Like porins, efflux pumps are also actively regulated by abundant global regulators as well as diverse intracellular or extracellular signals in addition to the local regulator. Transcription of the mexAB‐oprM operon is controlled by multiple regulatory proteins that recognize and bind to the promoter of mexAB‐oprM directly with or without the presence of specific signals. The MerR‐like regulator BrlR is required for the maximal expression of the mexAB‐oprM operon and the DNA binding assay revealed that BrlR activated the expression of mexAB‐oprM by directly binding to the promoter of the efflux operon (Liao et al., 2013). Expression of the mexAB‐oprM operon is influenced by the TCS CpxS/CpxR which may sense envelope stress generated by certain extracellular physical or chemical perturbations in P. aeruginosa (Tian & Wang, 2023). CpxR was found to positively regulate the expression of mexAB‐oprM by directly binding to the promoter of the efflux operon (Tian et al., 2016). Instead of activation, a LysR‐type transcriptional regulator PA3225 functions as a repressor that inhibits the expression of mexAB‐oprM by binding to the promoter (Hall Clayton et al., 2017). NalD is a TetR‐family repressor that inhibits the expression of mexAB‐oprM by targeting its promoter as well (Morita, Cao, et al., 2006). In clinical strains, mutations in nalD are frequently observed, which leads to the overexpression of mexAB‐oprM and the occurrence of multidrug resistance (Yan et al., 2019). Remarkably, the Zn²⁺‐responsive TCS CzcS/CzcR was recently found to repress the expression of mexAB‐oprM through the direct binding of CzcR to the promoter of the efflux operon (Chen et al., 2023). Additionally, the expression of mexAB‐oprM is repressed by the response regulator RocA2 which transduces signals from two sensor histidine kinases RocS1 and RocS2 (Sivaneson et al., 2011), and is activated in response to the quorum‐sensing signal C4‐HSL and the high concentration of Ca²⁺ (Khanam et al., 2017; Sawada et al., 2004).

Regulation of MexCD‐OprJ

MexCD‐OprJ was initially identified as a determinant of resistance to fluoroquinolones but is now known to export a wide variety of additional antibiotics including tetracyclines, quinolones, chloramphenicol as well as detergents, dyes, organic solvents, and so forth (Jamal et al., 2023; Masuda et al., 2000). In wild‐type P. aeruginosa strains, this efflux pump is normally quiescent but is inducible upon the presence of numerous chemicals such as benzalkonium chloride, chlorhexidine gluconate, tetraphenylphosphonium chloride, ethidium bromide, rhodamine 6G, and acriflavine (Figure 4) (Morita et al., 2001, 2003). The mexCD‐oprJ operon is regulated by two local repressors NfxB and EsrC which are located, respectively, upstream and downstream of the operon. NfxB shows high similarity to members belonging to the LacI‐GalR family and negatively regulates the mexCD‐oprJ operon as well as its own expression by binding to two 39‐bp repeats between the nfxB and mexC genes (Purssell & Poole, 2013; Shiba et al., 1995). Mutations in nfxB are frequently detected in clinical P. aeruginosa isolates, and these strains carrying nfxB mutations are known as nfxB‐type mutants which overproduce MexCD‐OprJ and display high levels of resistance to multiple antibiotics (Jakics et al., 1992; Poole, Gotoh, et al., 1996). The promoter of nfxB can also be recognized by the AraC‐family transcription factor VqsM. Loss of vqsM results in the repression of nfxB and an increased level of P. aeruginosa resistance to various antibiotics (Liang et al., 2014). As a homologue of NfxB, the second local repressor EsrC can bind to the promoter of the mexCD‐oprJ operon but regulate the expression of mexCD‐oprJ in the NfxB‐dependent manner (Purssell et al., 2015). Moreover, the expression of esrC is negatively regulated by NfxB (Purssell et al., 2015).

FIGURE 4.

The regulatory network of the efflux pump MexCD‐OprJ in Pseudomonas aeruginosa. NfxB and EsrC are local repressors of MexCD‐OprJ. The expression of mexCD‐oprJ is regulated by VqsM, AlgU (signal: envelope stress), LL‐37, sRNAs (spae3959.1, spae3706.1, and spae1558.1 induced by doxycycline), and many other compounds such as phenylethylamine, ethidium bromide, rhodamine 6G, chlorhexidine gluconate, acriflavine, benzalkonium chloride, dequalinium chloride, tetraphenylphosphonium chloride at the transcriptional level. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; IM: inner membrane; OM, outer membrane.

The expression of mexCD‐oprJ can be induced by envelope stress which is generally caused by membrane‐damaging agents. Induction of this operon by membrane‐damaging agents is mediated by the stress response sigma factor AlgU, which was initially identified as a regulator of alginate biosynthesis (Fraud et al., 2008; Martin et al., 1993). Interestingly, AlgU was also reported to reduce the expression of mexCD‐oprJ by upregulating the local repressor EsrC during envelope stress (Purssell et al., 2015). Doxycycline treatment upregulates sRNAs such as spae1558.1, spae3959.1, and spae3706.1, which further induce the expression of mexCD‐oprJ and the resistance of P. aeruginosa to doxycycline (Zhang et al., 2022). When P. aeruginosa cells are exposed to human host defence peptide LL‐37, the expression of the mexCD‐oprJ operon was upregulated, suggesting that the adaptive resistance of P. aeruginosa can be induced by host signals during infections (Strempel et al., 2013). By using a biosensor strain to indicate the expression level of mexCD‐oprJ and screening 379 metabolic compounds that act as carbon, nitrogen, phosphorus, and sulfur sources, phenylethylamine was found to be a novel inducer of the expression of the mexCD‐oprJ efflux operon (Muñoz‐Cazalla et al., 2023). Another study identified about 20 new compounds of which most were of relevance in clinical settings such as the disinfectant dequalinium chloride and the local anaesthetic agent procaine that can induce the expression of the mexCD‐oprJ operon by screening 240 compounds (Laborda et al., 2019).

Regulation of MexEF‐OprN

MexEF‐OprN extrudes antibiotics including chloramphenicol, fluoroquinolones, tetracycline, trimethoprim, and imipenem (Langendonk et al., 2021). In contrast to the local repressors for the MexAB‐OprM and MexCD‐OprJ efflux systems, expression of the MexEF‐OprN efflux pump is controlled by a local LysR‐like activator MexT which is encoded by the mexT gene located upstream of the mexEF‐oprN operon and upregulates the expression of mexEF‐oprN by binding to the nod box in the promoter region (Figure 5) (Köhler et al., 1999; Maseda et al., 2010). Similar to the MexCD‐OprJ efflux pump, MexEF‐OprM is normally quiescent in P. aeruginosa strains. Mutations in MexT activate the regulator from the inactive form to the active form, which leads to the overproduction of the MexEF‐OprN efflux pump and repression of the OprD porin, generating the so‐called nfxC‐type multidrug‐resistant mutants (Maseda et al., 2000). Most nfxC‐type mutants harbour disruptive mutations in an oxidoreductase‐encoding gene mexS which is located immediately upstream of the mexT gene and is also involved in controlling the expression of the mexEF‐oprN operon (Morita et al., 2015; Sobel Mara, Neshat, & Poole, 2005). MexS is regarded as a repressor of mexT and downregulates the expression of the mexEF‐oprN operon, while it was also interestingly found that disruption of mexS led to an increase in the concentration of electrophilic metabolites, activating MexT to induce the expression of mexEF‐oprN (Fargier et al., 2012).

FIGURE 5.

The regulatory network of the efflux pump MexEF‐OprN in Pseudomonas aeruginosa. MexT is a local activator of MexEF‐OprN. The expression of mexEF‐oprN is regulated by CmrA (signal: electrophilic stress), MexS, PA2048, ParS/ParR, RpoA, BrlR, MvaT, AmpR, MxtR, GcsR, oxidative stress, paerucumarin, cinnamaldehyde, Z‐ethylthio enynone at the transcriptional level. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; OM, outer membrane; IM, inner membrane.

In addition to activating the expression of mexAB‐oprM, BrlR activates the expression of mexEF‐oprN as well by directly binding to its promoter (Liao et al., 2013). RNA polymerase (RNAP) is an important target for the development of new antibiotics. It was found that a single amino acid substitution in RpoA, the α‐subunit of RNAP, resulted in a reduced expression level of the mexEF‐oprN operon (Cai, Lu, et al., 2023). RpoA can directly bind to the promoter of the mexEF‐oprN operon and a single amino acid substitution in RpoA led to a reduced affinity of binding to the target DNA sequence compared to the wild‐type RpoA, causing downregulation of the mexEF‐oprN operon (Cai, Liao, et al., 2023).

Transcriptomic analysis revealed that the expression of mexEF‐oprN genes was abolished in P. aeruginosa mutants with a defective TCS ParS/ParR, which plays a critical role in multidrug resistance (Wang et al., 2013). The orphan sensor kinase MxtR represses the expression of mexEF‐oprN by inhibiting the expression of mexT (Zaoui et al., 2012). In addition, the TyrR‐like Enhancer‐Binding Protein GcsR has been reported to inhibit the expression of the mexEF‐oprN operon (Lundgren Benjamin et al., 2013; Sarwar et al., 2016). AmpR, which was found to upregulate the expression of mexAB‐oprM, downregulates the expression of mexEF‐oprN (Balasubramanian et al., 2012). Analysis of in vitro‐selected chloramphenicol‐resistant clones led to the identification of a new class of MexEF‐OprN‐overproducing mutants carrying gain‐of‐function alterations in the AraC‐like transcriptional regulator CmrA, which upregulates mexT and mexEF‐oprN through MexS and PA2048 (Juarez et al., 2017). During electrophilic stress, accumulated reactive electrophilic species can induce the expression of mexEF‐oprN in the dependence of CmrA (Juarez et al., 2017). MvaT is an H‐NS‐family global regulator in P. aeruginosa that controls the expression of multiple virulence genes (Diggle Stephen et al., 2002). It was shown that deletion of mvaT led to a substantially increased expression level of the mexEF‐oprN operon in MexS or MexT‐independent manners and the mutant became more resistant to chloramphenicol and norfloxacin than its parent strain (Westfall et al., 2006).

Expression of mexEF‐oprN is also induced by several intracellular or extracellular signals. Pseudomonas aeruginosa harbours the operon of pvcABCD to produce paerucumarin and the abolishment of paerucumarin biosynthesis by mutating pvcB was found to cause increased sensitivity of P. aeruginosa to chloramphenicol and ciprofloxacin owing to the transcriptional repression of mexT and mexEF‐oprN genes in spite of the simultaneous repression of mexS in the pvcB mutant (Iftikhar et al., 2020). Cinnamaldehyde, the inducer of mexAB‐oprM, can induce the expression of mexEF‐oprN as well at the subinhibitory concentration (Tetard et al., 2019). Upon screening 3,280 compounds for inhibitors of quorum‐sensing‐regulated genes, a recent study identified a quorum‐sensing inhibitor Z‐ethylthio enynone that triggers the overproduction of the MexEF‐OprN efflux pump to alleviate stress induced by the inhibitor (Kristensen et al., 2024). Moreover, exposure of P. aeruginosa to chlorine led to an increased resistance to antibiotics due to the oxidative stress‐induced upregulation of the mexEF‐oprN efflux operon (Hou et al., 2019).

Regulation of MexXY

In contrast to other RND efflux pumps, the operon mexXY encoding components of the efflux pump MexXY‐OprM does not contain the gene encoding the outer membrane channel. It was found that MexXY formed a complete RND efflux pump together with OprM from the MexAB‐OprM efflux pump (Mine et al., 1999). MexXY‐OprM is recognized as one of the primary resistance determinants to aminoglycosides and also plays an important role in extruding other antibiotics such as tetracycline, erythromycin, and cefepime (Morita et al., 2012b). Remarkably, a unique gene oprA located downstream of the mexY gene was found to form an operon with mexXY and encode an outer membrane channel named OprA to form MexXY‐OprA in certain PA7‐like strains (Roy et al., 2010). OprA shares 47% of sequence identity with OprM, and some antibiotics such as bi‐anionic β‐lactams carbenicillin and sulbenicillin were identified to be specific substrates of the MexXY‐OprA efflux pump (Morita et al., 2012a; Singh et al., 2020). MexZ is a TetR‐family local repressor of the mexXY operon, which inhibits the expression of mexXY by directly binding to a 20‐bp palindromic sequence in the mexX‐mexZ intergenic region (Figure 6) (Matsuo et al., 2004). In clinical isolates, mutations such as code‐shift mutations, base deletion mutations, or point mutations are commonly found, which lead to the overproduction of MexXY, thereby increasing the resistance of P. aeruginosa to multiple antibiotics (Guénard et al., 2014).

FIGURE 6.

The regulatory network of the efflux pump MexXY in Pseudomonas aeruginosa. MexZ is a local repressor of MexXY. The expression of mexXY is also regulated by ArmZ, SuhB, Fmt, RplY, FolD, RplU, RpmA, PmrA/PmrB (signal: halogenated indoles), ParS/ParR (signal: Zn²⁺), AmgS/AmgR (signal: envelope stress), PA2798, PA5528, HtpX, PNPase, norspermidine, ribosome inhibitors, and oxidative stress at the transcriptional level. Grey arrow, transcription; black arrow, translation; red arrow, movement; solid pink arrow, direct upregulation by binding at the promoter; dashed pink arrow, indirect upregulation or upregulation with unclear molecular mechanisms; solid T‐shaped blue lines, direct downregulation by binding at the promoter; dashed T‐shaped blue lines, indirect downregulation or downregulation with unclear molecular mechanisms; IM, inner membrane; OM, outer membrane.

Expression of the mexXY operon is induced by ribosome inhibitors such as chloramphenicol, tetracycline, macrolides, and aminoglycosides (Jeannot et al., 2005). Under the treatments of ribosome inhibitors, stalling of the ribosome on PA5471.1 during the translation of newly synthesized mRNA triggers the transcription of a MexZ‐targeting anti‐repressor gene named armZ (Morita et al., 2009). ArmZ physically interacts with MexZ to reduce the binding ability of MexZ to target DNA, leading to the overexpression of the mexXY operon (Hay et al., 2013; Kawalek et al., 2019; Morita, Sobel Mara, & Poole, 2006; Yamamoto et al., 2009). Transcriptional expression of armZ is repressed by the TCS PmrA/PmrB which is activated by halogenated indoles (Dou et al., 2023). In addition, polynucleotide phosphorylase (PNPase) was found to control the translation of the armZ mRNA through its 5′ UTR (Fan et al., 2021). Therefore, mutations in the PNPase‐encoding gene upregulate the expression of the mexXY genes and increase the resistance of P. aeruginosa to aminoglycoside antibiotics (Fan et al., 2021). Furthermore, the expression of armZ is upregulated by oxidative stress, PA2798, and downregulated by suhB, fmt, folD, rplY, and rplU‐rpmA (Caughlan Ruth et al., 2009; El'Garch et al., 2007; Fraud & Poole, 2011; Genova et al., 2023; Lau Calvin et al., 2012; Shi et al., 2015).

Analysis of amikacin‐based selection of MexXY‐overproducing mutants led to the identification of the TCS ParS/ParR which positively regulates the expression of the mexXY operon independently of the repressor MexZ (Muller et al., 2011). In addition, Zn²⁺ was found to induce the expression of mexXY, and the increased expression of mexXY was also mediated by ParS/ParR (Poole et al., 2019). Mutation‐driven activation of PmrA/PmrB elevates the production of norspermidine, which leads to the upregulation of the mexXY operon and increases resistance to aminoglycoside (Bolard et al., 2019). Expression of the mexXY operon is also induced by AmgS/AmgR which is a TCS essential for aminoglycosides resistance in response to envelope stress (Krahn et al., 2012). AmgS/AmgR‐induced upregulation of mexXY is mediated by the cytoplasmic membrane‐associated protease HtpX and a membrane protein PA5528 (Lau et al., 2015).

INTRICATE REGULATION OF PORINS AND EFFLUX PUMPS IN P. aeruginosa

Antibiotic accumulation in bacterial cells is crucial for exhibiting their antibacterial activity. Bacteria, including P. aeruginosa, have evolved inducible behaviours to prevent the influx and activate the efflux of antibiotics by modulating the expression of porin proteins and efflux pumps. However, antibiotic resistance, particularly caused by reduced membrane permeability and active efflux, has a cost in nutrient uptake and bacterial fitness (Jordana‐Lluch et al., 2023; La Rosa et al., 2021). Thus, the downregulation of porins and upregulation of efflux pumps are transiently activated at the transcriptional or post‐transcriptional level when bacteria are exposed to antibiotics or other intracellular and extracellular signals.

Three major groups of regulators control the expression of porins and efflux pumps: local regulators, global regulators, and TCSs. Most RND efflux pump operons are found genetically linked to a local regulator such as MexR, NfxB, MexT, and MexZ as mentioned above. In many cases, local regulators are repressors that are transcribed divergently from the efflux operon, but some, such as MexT, can be activators that are normally in the inactive state. Thus, efflux genes are generally expressed at a low, basal level. Loss‐of‐function or gain‐of‐function mutations in local repressors or activators are frequently identified as key factors driving efflux pump overexpression and antibiotic resistance not only in clinical isolates of P. aeruginosa but also in isolates of other bacterial species such as E. coli, K. pneumoniae, and Salmonella enterica serovar Typhimurium (Olliver et al., 2004; Schneiders et al., 2003; Webber Mark et al., 2005). In addition to mutations, efflux pumps are often induced by binding a ligand, usually the substrate of efflux pumps, to the local regulator. For example, a variety of structurally dissimilar ligands have been found to bind to AcrR, the local regulator of AcrAB (Li et al., 2007). However, direct ligand binding to local regulators is rarely reported in P. aeruginosa. Instead, local repressors such as MexR and MexZ can be dissociated from the promoters of mexAB‐oprM and mexXY through protein–protein interactions with the anti‐repressors ArmR and ArmZ, respectively.

In addition to local regulators, porin proteins and efflux pumps are controlled by global regulators. A well‐studied example of global regulation is the highly conserved efflux pump AcrAB in Enterobacteriaceae (Wang‐Kan et al., 2021). AcrAB is directly regulated by three global regulators MarA, Rob, SoxS in E. coli, by RamA in Salmonella, and by RamA, RomA, RarA in Klebsiella (Colclough et al., 2020). Notably, most of these global regulators are members of the AraC/XylS family. The global regulation of porin and efflux genes in P. aeruginosa has also been intensively studied. A large number of global regulators have been identified that regulate the expression of porin and efflux genes either independently or in dependence of the local regulators. These global regulators are highly diverse and belong to different families, including the AraC/XylS family, the MerR family, the TetR family, the H‐NS family, as well as TyrR‐like Enhancer‐Binding proteins, and subunits of RNA polymerase.

Another important group of regulators controlling the expression of porin proteins and efflux pumps are TCSs. A typical TCS, consisting of a histidine kinase and a response regulator, plays a critical role in fine‐tuning the expression of target genes in response to environmental signals. P. aeruginosa has the highest number of TCSs compared to other bacterial species with over 60 TCSs being encoded by the genome (Rodrigue et al., 2000). Although the functions of most TCSs in P. aeruginosa are not fully characterized, increasing studies have shown that they are importantly involved in the direct or indirect regulation of porin and efflux genes. Some porin and efflux genes are located close to TCS genes, and these TCSs have been found to specifically regulate the neighbouring porin and efflux genes. For example, the TCS PhoP/PhoQ regulates the expression of the upstream porin gene oprH in P. aeruginosa, AdeS/AdeR is responsible for the expression of the adjacent efflux gene operon adeABC in Acinetobacter baumannii, and SmeS/SmeR is responsible for the expression of the adjacent efflux gene operon smeABC in Stenotrophomonas maltophilia (Li et al., 2002; Marchand et al., 2004). However, the TCSs found in P. aeruginosa act more as global regulators, such as ParS/ParR and CzcS/CzcR, that co‐regulate the expression of porin or efflux genes located in separate regions of the genome. The diversity of global regulators as well as the abundance and versatility of TCSs in P. aeruginosa further constitute an extraordinarily complicated regulatory network of porin and efflux genes, providing a critical explanation for why this pathogen is difficult to eradicate by antibiotic therapies and persists in various host and environmental niches.

ANTI‐RESISTANCE BY MODULATING ANTIBIOTIC INFLUX AND EFFLUX MACHINERIES

The functions, structures, and substrates of porin proteins and efflux pumps have been extensively studied for a long time, leading to the development of effective synthetic and semi‐synthetic adjuvants especially the efflux pump inhibitors (EPIs) which physically block the extrusion effects of efflux pumps. Unfortunately, none of these adjuvants are currently used in clinical antimicrobial therapies due to their instability, low selectivity, and cytotoxicity (Spengler et al., 2017). As summarized in this article, evidence is accumulating that the production of porin proteins and efflux pumps is tightly controlled by regulators that are either dependent on intracellular or extracellular signals or not. Isolation of natural anti‐resistance adjuvants to interfere with the abundant regulatory pathways provides more promising options for the development of novel antibacterial strategies by increasing the production of porins or reducing the production of efflux pumps with lower cytotoxicity and better tolerability.

Traditional Chinese medicine is a form of natural medicine characterized by high safety, remarkable efficacy, and low toxicity (Matos et al., 2021). Many active compounds extracted from traditional Chinese medicine have shown inhibitory effects on the expression of efflux pumps, showing the possibility of enhancing the antibacterial effects of antibiotics. For example, piperine, a kind of cinnamamide alkaloids extracted from the Chinese herb pepper, has been demonstrated as a novel expression inhibitor of the MexAB‐OprM efflux pump, and the combination of piperine and imipenem showed a synergistic effect on eliminating carbapenem‐resistant P. aeruginosa (Liu et al., 2023). Biomedical materials such as nanoparticles also exhibit great capability as adjuvants to potentiate the intracellular accumulation of antibiotics. After long‐term exposure of P. aeruginosa to platinum nanoparticles, it was found that oprD was upregulated while mexEF‐oprN was downregulated, facilitating the intracellular accumulation of antibiotics and reducing the resistance of P. aeruginosa to imipenem and ciprofloxacin (Zhou et al., 2023). Since platinum nanoparticles are widely utilized in medical applications (Mikhailova, 2022), the combination of platinum nanoparticles and traditional antibiotics provides a promising approach to combat antibiotic‐resistant P. aeruginosa. Probiotics are used as dietary supplements which provide multiple health benefits (Ali et al., 2023). It was recently shown that probiotic cell‐free supernatants significantly increase the expression of oprD and decrease the expression of several efflux operons such as mexAB‐oprM, mexCD‐oprJ, and mexEF‐oprN (Mehboudi et al., 2023), highlighting a therapeutical potential with the combination of probiotics and antibiotics.

CONCLUDING REMARKS

The management of P. aeruginosa infections remains a formidable challenge largely due to the ability of this pathogen to prevent the intracellular accumulation of antibiotics. Extensive research over the past decades has shown that the resistance of P. aeruginosa depends not only on the presence of the machineries responsible for antibiotic transportation but also on their expression. Although many excellent studies have not been included due to the space limitation, the examples summarized in this review have exhibited a very complicated regulatory network involving local regulators, global regulators, and TCSs that link the intracellular antibiotic accumulation to various intracellular and extracellular signals.

With the increased knowledge of the regulatory mechanisms of porins and efflux pumps, in recent years, attention has been given to explore the potential of natural compounds or existing biomedical materials to serve as safe and efficient adjuvants for common antibiotics to promote intracellular antibiotic accumulation and combat multidrug‐resistant P. aeruginosa. Although the major antibiotic resistance determinants in P. aeruginosa have been identified, much remains unknown about the regulation of these antibiotic resistance determinants. Studies are still needed to further explore inducers of intracellular antibiotic accumulation and to elucidate the regulatory mechanisms of antibiotic influx and efflux in P. aeruginosa. A deeper understanding of which signals trigger intracellular antibiotic accumulation and how antibiotic influx and efflux are modulated is pivotal for the development of novel therapeutic strategies.

AUTHOR CONTRIBUTIONS

Weiyan Wu: Writing – original draft. Jiahui Huang: Writing – original draft. Zeling Xu: Conceptualization; writing – review and editing.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (Nos 32370188 and 32100020), and the Guangdong Basic and Applied Basic Research Foundation (Nos 2023A1515012775 and 2022A1515010194).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest with the contents of this article.

Wu, W. , Huang, J. & Xu, Z. (2024) Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications. Microbial Biotechnology, 17, e14487. Available from: 10.1111/1751-7915.14487