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. 2024 Apr 29;19(9):771–782. doi: 10.2217/fmb-2023-0272

Efflux pump inhibitor chlorpromazine effectively increases the susceptibility of Escherichia coli to antimicrobial peptide Brevinin-2CE

Cao Bing 1,‡, An Mengjuan 1,‡, Ma Xinyu 1, Zhu Chixin 1, Tan Xinyao 1, Sun Yan 1,*, Li Zhi 1,**
PMCID: PMC11290751  PMID: 38683168

Abstract

Aim: The response of E. coli ATCC8739 to Brevinin-2CE (B2CE) was evaluated as a strategy to prevent the development of antimicrobial peptide (AMP)-resistant bacteria. Methods: Gene expression levels were detected by transcriptome sequencing and RT-PCR. Target genes were knocked out using CRISPR-Cas9. MIC was measured to evaluate strain resistance. Results: Expression of acrZ and sugE were increased with B2CE stimulation. ATCC8739ΔacrZ and ATCC8739ΔsugE showed twofold and fourfold increased sensitivity, respectively. The survival rate of ATCC8739 was reduced in the presence of B2CE/chlorpromazine (CPZ). Combinations of other AMPs with CPZ also showed antibacterial effects. Conclusion: The results indicate that combinations of AMPs/efflux pump inhibitors (EPIs) may be a potential approach to combat resistant bacteria.

Keywords: : AMP, B2CE, bacterial resistance, CPZ, EPI

Plain language summary

Summary points.

  • The sub-lethal concentration of B2CE resulted significant changes in the expression levels of numerous genes in E. coli.

  • The deletion of acrZ and sugE proved that these efflux pumps are responsible for pumping B2CE out of the cells, weakening its inhibitory effect on E. coli.

  • The combination of B2CE and CPZ increased the antibacterial effect on E. coli by fivefold, which verified the combined use of EPI achieves effective antibacterial effects at lower peptide concentrations, thus reducing the risk of bacterial resistance to B2CE.

  • The B2Ka and P2CE combined with CPZ also increased the antibacterial effects on E. coli, which suggested that the combined use of amphibian AMPs and EPIs is a feasible future clinical application strategy.

Antimicrobial peptides (AMPs), found in all known forms of life, have gained increased interest as potential therapeutic options for the treatment of various diseases [1,2]. As antibiotic resistance continues to be an increasing problem worldwide, AMPs are a potential option that inhibit antibiotic-resistant pathogens [3]. However, despite the low risk of resistance development to AMPs, there have been reports of microbial pathogens that have evolved mechanisms to withstand AMPs. Certain bacteria show the inherent ability to resist AMPs, while most other microorganisms can develop countermeasures such as the activation of stress response systems and/or efflux systems, modification of the cell envelope and/or extracellular facet of the cytoplasmic membrane, modification of intracellular targets and the expression of proteases/peptidases specifically cleaving AMPs [4]. Thereby, the combined use of AMPs with other agents, such as AMP-AMPs, AMP-antibiotics and AMP-efflux pump inhibitors (EPI), provides a new direction to overcome the drawback of monotherapies during infections [5,6]. For example, the AMPs CAP18 and BMAP-27 have shown a synergistic effect with NMP, PaβN and CCCP against multidrug-resistant Staphylococcus aureus (MRSA) isolates [7]. However, not all EPIs have a synergistic effect with AMPs; in the same study, it was found that the combination of LL37, CRAMP and BMAP-28 with NMP, PaβN and CCCP, respectively, did not show any effect. Thus, the combination of AMP-EPIs as an alternative treatment option needs further study.

According to the data from ADP3 (https://aps.unmc.edu/, August 2023), 1196 of a total 3569 active AMPs come from amphibians, making them the most abundant source. Among the 12 well-established AMP families, there are 94 records in ADP3 of peptides from the brevinine-2 family, which are isolated from only Eurasian frogs [8]. Brevinin2-CE (B2CE), belonging to the brevinin-2 family, was previously isolated in our lab from the skin secretions of Rana chensinensis and has exhibited antibacterial effects [9]. The combination of B2CE with several antibiotics and the modified peptides derived from B2CE have been employed to treat different infections, including even multidrug-resistant E. coli and S. aureus strains [10,11]. However, some pathogens have been reported as resistant to amphibian AMPs. For example, Pexiganan, an analog of Magainin that was originally isolated from Xenopus leavis, was used to treat mild infections associated with diabetic foot ulcers. After long-term exposure to Pexiganan, Pseudomonas fluorescens and E. coli displayed resistance to the AMP [12]. Also, S. aureus exposed to Pexiganan developed resistance not only to Pexiganan but also to Melittin, an AMP derived from bees [13]. It is necessary to study the mechanism of action of each AMP and the stress response of bacteria against AMPs, to achieve the best therapeutic effect and minimal side effects when using AMPs in clinical treatments.

In this paper, we have investigated B2CE's potential and strategies for preventing the development of AMP-resistant E. coli to shed light on how we might control the development of resistant bacteria. E. coli is a well-characterized model organism, as well as a common cause of urinary tract infections, gastrointestinal infections and other diseases in humans. Its ability to develop resistance to antimicrobial peptides has been researched extensively [12], hence it's choice as a model organism here.

Materials & methods

Strains & AMPs

E. coli ATCC 8739 (also numbered as CICC 10302) was purchased from China Center of Industrial Culture Collection (Beijing, China). Brevinin-2CE (B2CE), Brevinin-2Ka (B2Ka) and Palustrin-2CE (P2CE) peptides were synthesized by Wuhan Moore Biotechnology Co., Ltd (Wuhan, China).

Sample processing

E. coli ATCC 8739 was inoculated in MH medium at 37 °C. When the OD600 reached 0.5 (about 2 h after inoculation), B2CE peptide was added into the medium to final concentrations of 0.5 μmol/l, 0.75 μmol/l, and 1.5 μmol/l, respectively. Also, the untreated cells were used as control. The test and control groups were grown and the cell numbers were calculated every 30 min. The growth curve of each sample was drew.

Transcriptome sequencing

E. coli treated by 0.5 μmol/l B2CE peptide for 40 min was picked as the experimental group, while the cells untreated was used as control. Total RNA was isolated using Qiagen RNeasy Mini Kit (Hilden, Germany) and divided into two portions. One portion of the extracted RNA was sent to Shanghai Parsono Biotechnology Co., Ltd. (Shanghai, China) for transcriptome sequencing. The other portion were stored for real-time PCR assay.

Real-time quantitative PCR

According to the transcriptome sequencing results, several candidate genes showing obvious changes of expression-level were selected for further real-time PCR analysis. The primers used were summarized in Supplementary Table 1. The stored RNA sample were took out and the abundant rRNA from the total RNA was removed with Ambion MICROBExpress kit (MA, USA). RNA was reverse-transcribed into cDNA using TaKaRa PrimeScript RT reagent Kit (Dalian, China) with gDNA Erase. Real-time PCR was carried out with TaKaRa SYBR Premix Ex Taq II on CFX96 touch real-time PCR detection system (Bio-Rad, USA).

Construction of gene knock-out strains

The knock-out strains of acrZ and sugE were constructed using a two-plasmid-based CRISPR-Cas9 system. The sgRNA and primers used in this study are given in Supplementary Table 2. pTargetF-sgRNA-acrZ and pTargetF-sgRNA-sugE were constructed as described previously [14]. And the donor DNA was synthesized by overlap PCR. E. coli ATCC 8739 harboring pCas was prepared. Then 100 ng of pTargetF series DNA and 400 ng of donor DNA were co-electroporated. Cells were recovered at 30 °C for 1 h, and then spread onto LB agar containing kanamycin (50 mg/l) and spectinomycin (50 mg/l) and incubated overnight at 30 °C. Transformants were identified by colony PCR and DNA sequencing. For the curing of pTargetF series, the edited colony harboring both pCas and pTargetF series was inoculated in LB medium containing kanamycin (50 mg/l) and IPTG (0.5 mmol/l). The culture was incubated for 8–16 h, diluted, and spread onto LB plates containing kanamycin (50 mg/l). The colonies were confirmed as cured by determining their sensitivity to spectinomycin (50 mg/l). pCas was cured by growing the colonies overnight at 37 °C nonselectively. Finally, the knockout strains ATCC8739ΔacrZ and ATCC8739ΔsugE were verified by sequencing.

Construction of refilled strains

The full coding region of acrZ and sugE were amplified and cloned into pSTV29 to construct the gene expression vectors. The primers used in this study are given in Supplementary Table 2. Then the recombinants were transformed into knockout strains ATCC8739ΔacrZ and ATCC8739ΔsugE, respectively, to obtain the refilled strains C-ATCC8739ΔacrZ and C-ATCC8739ΔsugE. Also, pSTV29 plasmid had been transformed into wild ATCC8739, ATCC8739ΔacrZ and ATCC8739ΔsugE to screen the control stains ATCC8739-pSTV29, ATCC8739ΔacrZ-pSTV29 and ATCC8739ΔsugE-pSTV29.

Antimicrobial assays

The minimal inhibitory concentration (MIC) of different test strains against B2CE were determined using a standard method as described previously [11]. Briefly, the knock-out, complementary, and refilled strains were cultured in MH liquid medium to the logarithmic phase and diluted to 1 × 106 cfu/ml. According to the previous experiments in the laboratory, the MIC value of wild-type E. coli ATCC 8739 against B2CE was 3 μmol/l. Thus B2CE was diluted with MH liquid medium to 12 μmol/l, 6 μmol/l, 3 μmol/l and 1.5 μmol/l, respectively. Then 100 μl of the AMPs and 100 μl of bacterial culture were mixed, respectively, and incubated for 24 h with shaking at 200 r/min. A mixture of 100 μl of MH medium and bacterial culture was negative control. The absorbance at 595 nm of different mixtures was detected on SpectraMax ABS plus microplate reader (MD, USA), and the MIC value was calculated. The examinations were repeated three times.

Bacterial survival rate assay

The wild-type E. coli and the knock-out strains were treated with B2CE the same as the above MIC assay, while the final concentration of B2CE in each group is 1.5, 0.75 and 0.37 μmol/l, respectively. The group without B2CE was the negative control. After incubation at 37 °C for 24 h, the cell numbers were counted using plating method. The cell numbers of the negative control was defined as 100% survival rate. The examinations were repeated three-times.

Combination effect of B2CE & other AMPs with chlorpromazine

B2CE was diluted with MH liquid medium to 3 μmol/l and 1.5 μmol/l, respectively. E. coli ATCC 8739 was cultured as previously and diluted to 1 × 106 cfu/ml. Then 100 μl of the culture was mixed with 100 μl B2CE-MH medium, and the final concentration of B2CE in each group is 1.5 μmol/l and 0.75 μmol/l respectively. At the same time, the bacterial EPI chlorpromazine (CPZ) was diluted using the above B2CE-MH medium to the final concentration 50 μg/ml. Then 100 μl of the culture was mixed with 100 μl CPZ-B2CE-MH medium. After incubation at 37 °C for 24 h, the survival rate was calculated as previously. The examinations were repeated for three-times.

The combination effect of other two AMPs, B2Ka and P2CE, with CPZ were evaluated following the same strategy. The final concentration of B2Ka and P2CE was 1.5 μmol/l and 6.25 μmol/l, respectively, according to previous studies, and the concentration of CPZ was 25 μg/ml.

Results

Effect of B2CE on the growth of E. coli ATCC 8739

Bacterial growth was inhibited when treated with 1.5 μmol/l and 0.75 μmol/l of B2CE. At 0.5 μmol/l, bacterial growth was affected slightly at first, but growth recovered quickly compared with the control group (Figure 1). One hour after treatment (3 h of bacterial growth), the total number of bacteria in the group treated with 0.5 μmol/l was similar to the control. At 1.5 h (total 3.5 h bacterial growth), growth was similar to the control group. According to these results, we treated E. coli ATCC 8739 in the following experiments with 0.5 μmol/l B2CE for 40 min.

Figure 1.

Figure 1.

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Growth curve of E. coli treated with different concentrations of B2CE.

Data are means ± standard deviations of at least three experiments.

Transcription profile of E. coli ATCC 8739 treated with B2CE

A gene was considered significantly regulated when the expression ratio (ratio of control cells/treated cells) was ≤0.5 (up-regulated) or ≥2.0 (down-regulated). Treatment of B2CE caused substantial changes in the transcription of 37 bacterial genes. Among these, the transcription of 35 genes was induced and two genes were repressed (Supplementary Table 3).

GO enrichment analysis was performed using the 37 differentially expressed genes. In the biological process, the expression of genes associated with translation and transport functions changed dramatically. In the cellular components, genes linked with organelles and plasma membranes showed expression level changes, most of which were ribosome-related genes and membrane-related genes. In the molecular function group, the expression of genes relating to structure molecular activity changed significantly. Based on the results of KEGG enrichment analysis, environmental signal processing is an important group of interest, with the pathways involved in membrane transport and signal transduction showing changes.

Thirty-five up-regulated genes were organized into different groups based on their functional annotations and the wider literature. The first included ratA, ecnB, yicR, ihfA and some ribosomal protein genes: ratA inhibits protein translation by binding to the 50S subunit; ecnB belongs to the entericidin family, which is related to bacteriolysis function; yicR is directly related to DNA repair; and ihfA is related to DNA integration. The second group contained many synthetic genes such as thrL and glnB, which are involved in amino acid synthesis, fsaB, which is involved in sugar synthesis, and mraY, which plays a role in peptidoglycan synthesis. The third group consisted of several transport-related genes such as ccmC, which transports ferroheme for cytochrome c synthesis, uphA, which transports six-carbon sugars, and tatC, which is involved in the translocation of a subset of proteins. Other members of this group included exbD and exbB, responsible for energy transmission, sugE, over-expression of which is linked to resistance to a variety of toxic substances, and acrZ, an efflux pump protein involved in drug efflux. Group four consisted of stress protein genes, such as cspB, cspC and cspE of the cold shock protein family, and bsmA, a stress protein related to the biofilm response to oxidative stress. The last group of up-regulated genes were endometrial protein genes, but the specific functions of these genes have not been reported.

The two down-regulated genes were srlE and uspA: srlE is part of the E. coli PTS system and participates in group transposition, which affects the transport of a substrate from the outside to the inside of the cells; uspA encodes a universal stress protein that protects against substances that may cause DNA damage.

Verification of the gene expression by real-time quantitative PCR

Several genes were selected from each group of the differentially expressed genes described above to verify the expression level by RT-PCR (Figure 2). The expression level of ecnB, yicR, mraY, UphA, sugE, acrZ, cspE and bsmA were up-regulated significantly compared with the control gene. srlE and uspA were down-regulated. These results were consistent with the results of transcriptome sequencing. The expression levels of three genes detected by RT-PCR, however, contradicted the results of the transcriptional profile. The ihfA gene was identified as being up-regulated by transcriptome sequencing, but its expression levels were unchanged when measured by RT-PCR. frdD and exbD were also identified as being up-regulated by transcriptome sequencing, but were down-regulated in RT-PCR.

Figure 2.

Figure 2.

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Validation of gene expression changes after B2CE stimulation by real-time quantitative PCR.

The expression level of gapA was set to be 1 to calculate the transcript change of other genes before and after B2CE treatment.

*p < 0.05.

Deletion of acrZ & sugE resulted in an increased sensitivity of E. coli ATCC 8739 against B2CE

Gene knockout strains of UphA, sugE and acrZ were constructed to investigate the functions of these genes under AMP stimulation. The antimicrobial assays indicated that except UphA (data not shown), sugE and acrZ would affect the MIC value of E. coli ATCC 8739 against B2CE (Figure 3A). Compared with the wild-type ATCC 8739, both the MIC values of acrZ and sugE gene knockout strains were decreased. The MIC value of ATCC8739ΔacrZ was decreased twofold and ATCC8739ΔsugE was decreased fourfold, indicating that gene knockout strains were more sensitive to B2CE. The MICs of wild strains and gene knockout strains transformed with the pSTV29 plasmid did not change, indicating that the plasmid vector did not affect the experimental results. Re-introduction of acrZ and sugE genes into the knockout strain caused the MIC of the refilled strains to return to a normal value, as compared with the wild ATCC 8739 type.

Figure 3.

Figure 3.

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MIC values (A) and survival rate (B) of each strain against B2CE.

ATCC 8739 was a wild-type strain of E. coli, ATCC8739ΔacrZ and ATCC8739ΔsugE were acrZ and sugE gene knockout strains, respectively. ATCC8739-pSTV29, ATCC8739ΔacrZ-pSTV29 and ATCC8739ΔsugE-pSTV29 were transformed into plasmid control strains, respectively. C-ATCC8739ΔacrZ and C-ATCC8739ΔsugE were acrZ and sugE gene complementation strains, respectively. The survival rate of untreated sample of each strains was set to be 100%. The significant difference between test strain and the relative untreated sample of each group was calculated.

*p < 0.05.

The survival rates of wild and knockout strains were calculated after treatment with different concentrations of B2CE. After treatment with AMP, the survival rates decreased compared with the untreated control, no matter the wild strain or the knockout strains (Figure 3B). Following treatment with 1.5 μmol/l of B2CE, the survival rates of ATCC8739ΔacrZ and ATCC8739ΔsugE were about 8.5% and 1.8%, respectively, which is much lower than the survival rates of wild strain ATCC 8739. Treatment with 0.75 μmol/l resulted in survival rates of ATCC8739ΔacrZ and ATCC8739ΔsugE that were slightly increased compared with 1.5 μmol/l, but still much lower than that of the wild-type. The deletion of acrZ and SugE genes resulted in increased sensitivity of knockout strains to B2CE.

Enhanced efficacy of EPI/AMP combination treatments versus E. coli ATCC 8739

CPZ, a classical EPI, was used to inhibit efflux pump activity. So, the sensitivity of E. coli to B2CE was evaluated. When treated with CPZ combined with 1.5 μmol/l B2CE, the survival rate of wild ATCC 8739 was about 5%, which is much lower than the survival rate (approximately 25%) of cells treated with the same concentration of AMP only. A similar pattern was observed between the cells treated with 0.75 μmol/l B2CE only and 0.75 μmol/l B2CE/CPZ combination, where the survival rate was 41% in the AMP only group and 8% in the combined group (Figure 4). When treating E. coli with B2CE and CPZ, CPZ might help to reduce the bacterial survival rate about fivefold. For ATCC8739ΔacrZ and ATCC8739ΔsugE, there were no obvious differences in the survival rates between the AMP-only group and the combined B2CE/CPZ group.

Figure 4.

Figure 4.

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The survival rate of wild and gene deletion strains against B2CE combined with EPI CPZ.

Each strain was divided into two groups. One group was treated with B2CE alone, and the other group was treated with B2CE and EPI CPZ simultaneously. The survival rate of untreated sample of each strains was set to be 100%. Different letters on the top of each column indicate the significant difference.

In addition to B2CE, we also tested B2Ka and P2CE, two other amphibian AMPs. B2Ka was isolated from Rana kukunoris and P2CE from R. chensinensis [9]. When B2Ka/P2CE were used alone, the survival rate was greater than 70%, and the bacteriostatic effect was not obvious. When treated with B2Ka/CPZ in combination, the survival rate decreased to 52%. When treated with P2CE/CPZ, the survival rate of E. coli decreased further to less than 5% (Figure 5). The combination of AMPs and CPZ seems to drastically improve the antibacterial effect.

Figure 5.

Figure 5.

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The survival rate of ATCC 8739 against B2Ka and P2CE combined with CPZ.

ATCC 8739 was treated with AMPs only or combined with CPZ. The survival rate of untreated sample was set to be 100%. Different letters on the top of each column indicate the significant difference.

Discussion

The mechanism by which AMPs inhibit pathogens is different from antibiotics, so initially, it was believed that bacteria were not likely to develop resistance to AMPs. However, research has shown that after continuous cultivation in a medium containing AMPs for 600–700 generations, the tested E. coli and P. aeruginosa strains have developed resistance to AMPs [12]. The risk of inducing the development of bacteria resistant to AMPs in clinical applications makes the study of AMP resistance mechanisms important. In their natural state, most bacteria are exposed to sub-lethal concentrations of AMPs [15]. This study aimed to identify what are the effects of sub-lethal concentrations of AMPs on the genetic and physiological characteristics of host bacteria, and whether they are similar to antibiotics in promoting bacterial resistance.

Multiple studies have shown that after stimulation with sub-lethal concentrations of AMPs, the genetic expression of host bacteria strains markedly changes [16]. For example, after stimulation with Bac7(1–35), the expression of 70 genes in E. coli changed, with 26 down-regulated and 44 up-regulated [17]. Cecropin A stimulation led to changes in the expression levels of 26 genes in E. coli, with three up-regulated and 23 down-regulated [18]. In this study, after treatment with sub-lethal concentrations of B2CE, 35 genes were up-regulated and two genes were down-regulated. Together, these findings suggest that E. coli utilizes different stress responses to withstand exposure to different AMPs. Genes down-regulated by Bac7 (1–35) were generally involved in energy metabolism, carbon metabolism, amino acid metabolism and transport proteins located in the cell membrane, while up-regulated genes were generally involved in cell structure synthesis, transport proteins, and protein synthesis and modification. Similarly, for Cecropin A, the down-regulated genes were involved in cell structure synthesis, energy metabolism and DNA repair, as well as some with unknown functions, while the up-regulated genes were involved in RNA transcription and processing, amino acid synthesis and metabolism. For B2CE, the up-regulated genes were involved in sugar metabolism and energy metabolism, protein synthesis and modification, transport and signal transduction. Of the two down-regulated genes, one was a PTS transport system protein and the other was an emergency protein.

Further real-time PCR assays validated the changes in gene expression of most candidate genes screened from the transcriptome. For example, yicR is related to DNA repair [19], and mraY is involved in the synthesis of peptidoglycan [20]. The obvious up-regulation of these two genes is speculated to be related to cellular repair after bacterial damage. Both cspE and bsmA are stress protein genes [21,22], and the alteration of these two genes might be the result of the response to B2CE stimulation. UphA, sugE and acrZ are transport-related protein genes and may be involved in the host's resistance to antibiotics [23–25]. The relationship between the up-regulation of these genes and E. coli stress ability after B2CE stimulation deserves further studies.

Interestingly, the expression levels of three genes detected by RT-PCR were contradictory to initial transcriptome studies. The protein encoded by ihfA gene is one of the two subunits that constitute the integration host factor (IHF). IHF is a nucleoid-associated protein that functions as a global virulence regulator in E. coli [26]. At present, it seems that the ihfA gene is not directly related to the response of E. coli to AMP stimulation, so the result of real-time quantitative PCR is more reasonable in this study. Although frdD V111D confers ampicillin resistance in E. coli BW25113 [27], the main function of frdD is the conversion of fumaric acid to succinic acid, which is an important reaction in the tricarboxylic acid cycle. The down-regulation of frdD may be due to the inhibition of bacteria energy metabolism as the cells may be damaged after AMP treatment. This is consistent with observations that the growth of the bacteria is inhibited. ExbD forms transmembrane complexes with TonB (also known as ExbA) and ExbB transports some specific substrates, such as vitamin B12 [28]. The down-regulation of these genes may be due to the damage of the cell membrane by the AMP, which further affects the function of transport complexes.

It is tentatively inferred that sub-lethal concentrations of B2CE to some extent disrupt cell structure and affect normal cell function, thereby causing the cell to compensate by synthesizing structural proteins, which involves energy metabolism and substance transport. For example, mraY encodes a key enzyme in peptidoglycan synthesis, and the upregulation of mraY after B2CE stimulation is likely a compensatory response to cell wall damage caused by the AMP. FsaB catalyzes the cleavage of fructose-6-phosphate, a key enzyme in sugar metabolism, and FrdD reduces fumarate to succinate, a key enzyme in the TCA cycle, both of which play important roles in energy metabolism. Additionally, stimulation with B2CE also leads to changes in the expression of some emergency protein genes, such as the downregulation of uspA and the upregulation of cspC. It has been reported that the expression of uspA is influenced by carbon deficiency, heat stress, ROS, metal ions, ethanol, antibiotics and other stimulants [29]; similarly, in addition to being associated with cold shock adaptation, cspC also participates in heat shock, acid and oxidative stress response [30]. This indicates that the changes in emergency protein genes are a non-specific response of the host bacteria to B2CE stimulation.

Bacteria develop resistance to antibiotics in various ways, one of the most important being the use of efflux pumps to pump drugs out of the cell [31]. After stimulation with sub-lethal concentrations of B2CE, the expression of two efflux pump genes, acrZ and sugE, was up-regulated in E. coli. Experimental data has shown that acrZ and acrAB are both influenced by upstream regulators such as marA, rob and soxS, and that the absence of acrZ leads to increased sensitivity of E. coli to various antibiotics, such as tetracycline, puromycin and chloramphenicol [2,25]. Treatment of E. coli with duloxetine and/or chloramphenicol led to an upregulation of acrZ expression, resulting in resistance of the host bacteria to 12 kinds of antibiotics [32]. SugE, recently renamed as Gdx, has been shown in several studies to mainly transport small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds [33]. With further research, it has been found that sugE causes multidrug resistance in host bacteria, such as E. coli, Enterobacter cloacae, Acinetobacter baumannii as well as some species of Salmonella and Shigella [34,35,36,37]. In this study, after knocking out acrZ and sugE in E. coli, both ATCC8739ΔacrZ and ATCC8739ΔsugE showed increased sensitivity to B2CE, with MIC values decreasing by twofold and fourfold, respectively. Also, after the treatment with the same concentration of B2CE, the survival rate of ATCC8739ΔacrZ and ATCC8739ΔsugE was lower than that of the wild strain. These results suggest that acrZ and sugE play a role in the host bacteria's resistance to the AMP B2CE, and it is speculated that this may be related to their efflux function.

Considering the important role of efflux pumps in bacterial resistance, the long-standing focus of research has been on the use of EPIs to restore the antibacterial effects of existing antibiotics. Currently, there are seven families of efflux pumps in bacteria, including RND, ABC, MFS, SMR, MATE as well as two newly reported families, PACE and AbgT. Among them, the RND family is only present in Gram-negative bacteria, and AcrAB-TolC of the Enterobacteriaceae family is a well-studied representative of the RND family. AcrZ is an accessory protein in AcrAB-TolC. The SMR family members include EmrE, SugE and others. Research suggests that SugE originated from an ancient and conserved guanidinium ion efflux pump [38]. CPZ is a phenothiazine drug, mainly used for the treatment of mental diseases, but it has also been shown to inhibit the AcrAB-TolC efflux pump [39]. It has been found in many bacteria including E. coli, S. aureus, Klebsiella pneumoniae, Mycobacterium intracellulare and P. aeruginosa that CPZ can reduce the MIC of antibiotics [31]. Our experiment confirmed that, after the deletion of acrZ, the abnormal function of AcrAB-TolC was not affected by the addition of CPZ, and it no longer influenced the sensitivity of the host bacteria to B2CE. However, for the wild-type E. coli, the use of CPZ made the host bacteria more sensitive to B2CE, resulting in decreased survival rate. This suggests that a strategy similar to the combined use of CPZ and antibiotics could also be considered for practical application in using EPIs and AMPs in combination. For example, researchers found three AcrAB-TolC pump inhibitors, EPM30, EPM35 and EPM43, which significantly enhanced the inhibitory effect on Salmonella when used in combination with the human AMP LL37 [40]. Polymyxin E (colistin) and polymyxin B are cyclic peptides synthesized from non-ribosomal synthetase of Paenibacillus polymyxa subsp. Colistinus that possess strong antimicrobial activity. Polymyxin E has good resistance to Gram-negative bacteria, while polymyxin B is not only resistant to Gram-negative bacteria, but also to some Gram-positive bacteria. However, with the commercial application of polymyxin E (colistin) and polymyxin B, an increasing number of microorganisms have developed resistance to polymyxins [41]. Research has attempted to restore the activity of existing drugs through the combined use of EPIs and polymyxins. CCCP, as an EPI, can effectively improve the resistance of intrinsically colistin-resistant bacteria to colistin, such as Proteus spp., Serratia marcescens, Morganella morganii, Bacteroides intermedia and Providencia spp. [42,43]. Studies have also found that novel plant-derived EPIs can be used in combination with AMPs. For example, curcumin inhibits the efflux pump AcrB, reducing the MIC of colistin to <2 μg/ml in Enterobacteriaceae [44]. Baicalin combined with EDTA can also inhibit the expression of efflux pump genes such as acrB, acrD, and tolC, thereby restoring the sensitivity of colistin-resistant Salmonella to colistin [45].

In this study, based on the combined use of B2CE and CPZ, attempts were made to use the AMPs B2Ka and P2CE in combination with CPZ. The results showed a significantly reduced survival rate in the combined group, indicating that the combination of AMP and EPI has a better inhibitory effect. This suggests further that the combination of an AMP/EPI is an effective strategy against the development of bacterial resistance.

Conclusion

In this experiment, transcriptome sequencing was used to identify changes in gene expression levels in the presence of B2CE. Many genes related to sugar metabolism, energy metabolism, protein synthesis and modification, transport, and signal transduction showed altered expression levels. Subsequently, real-time quantitative PCR was used to validate the expression levels of some typical genes, especially acrZ and sugE, the genes encoding for the efflux pump proteins. Gene knockout experiments revealed that the deletion of acrZ and sugE resulted in a twofold and fourfold decrease in the MIC values of E. coli in the presence of B2CE. Moreover, the survival rate of the knockout strains was lower than that of the wild-type strain. These data indicate that the efflux pumps of which acrZ and sugE are a part may be linked to pumping B2CE out of the cell, thus weakening the inhibitory effect of AMPs on E. coli. Furthermore, the experiment demonstrated that the combination of B2CE with CPZ resulted in a fivefold increase in the inhibitory effect on E. coli compared with the control group without the combined use of CPZ. Additionally, two other amphibian AMPs, B2Ka and P2CE, showed enhanced inhibitory effects on E. coli when used in combination with CPZ. This experiment is the first to report on the feasibility of combining amphibian AMPs with EPIs. It also suggests that the combined application of AMPs and EPIs is a viable strategy for reducing AMP resistance in the future.

Unfortunately, the mechanism of action of the synergistic effect of AMPs and EPIs requires further research. Specifically, investigations into the physical interactions between AMPs and efflux pumps/transport proteins could provide valuable information to guide the design of AMPs that can avoid efflux pumps and/or intracellular protein degradation. Furthermore, this structural information would be helpful in the design of EPIs to block efflux pump channels. In short, enhancing the antagonistic effects of existing AMPs on pathogenic microorganisms through the use of EPIs is an effective strategy in the fight against increasingly resistant microorganisms.

Supplementary Material

Supplementary Tables S1-S3
IFMB_A_2339751_SM0001.zip (46.6KB, zip)

Acknowledgments

Thanks to Wu Y for data analysis. Thanks to Cheng L and Hao Y for assistance with the RT-PCR and knock-out experiment. Thanks to Zhang XL for assistance with the MIC analysis.

Funding Statement

This work was supported by the Fundamental Research Funds for the Central Universities (GK202302003) and the Innovative Experiment Projects of Educational Ministry of China for Undergraduate (S202310718118).

Author contributions

Cao B and An MJ were co-first authors. Cao B is responsible for the majority of the experimental research and writing the initial draft. An MJ is responsible for the majority of the experimental research and collecting experimental data. Ma XY is responsible for data analysis. Zhu CX and Tan XY are responsible for some of the experimental operations. Sun Y is responsible for the finalization of the paper writing. LI Z is responsible for the experimental design.

Financial disclosure

This work was supported by the Fundamental Research Funds for the Central Universities (GK202302003) and the Innovative Experiment Projects of Educational Ministry of China for Undergraduate (S202310718118). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

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Supplementary Materials

Supplementary Tables S1-S3
IFMB_A_2339751_SM0001.zip (46.6KB, zip)

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