The imperative need for developing innovative natural antimicrobial drugs is emphasized by the challenges posed by AMR15,16. RJ, has been well known for its potential health benefits by exhibiting a broad spectrum of antibacterial activities3,4. Notably, it has garnered attention for ability to combat antibiotic resistance in AMR P. aeruginosa17, positioning it as a promising candidate for novel natural antibiotics.
Transcription and translation represent the fundamental processes responsible for transferring the genetic information stored in DNA. In bacteria, transcription is facilitated by the RNAP core enzyme, composed of α2ββ’ω subunits. Given the vital role of transcription and the high conservation of RNAP in the bacterial realm, the discovery of antibacterial agents targeting on RNAP has been a focus since the mid-20th century18. A plethora of compounds, whether synthetic or isolated from microorganisms, have been identified for their ability to modulate transcription by inducing structural conformational changes in the RNAP core enzyme through specific site binding. Notable examples include Rifampicin, Streptolydigin, Fidaxomicin and Lipiarmycin19. Furthermore, certain compounds act on TFs associated with RNAP, thereby interfering with their binding to RNAP. Instances involve Bicyclomycin and the SB series19. A significant hurdle in the clinical development of many tested compounds is the frequent emergence of bacterial resistance due to mutations in their binding sites 20. Currently, only Rifampicin and Fidaxomicin/Lipiarmycin have received approval for market use. In contrast to the aforementioned drugs, which primarily target transcription processes by inducing structural changes in RNAP or its associated factors, our results demonstrate that RJ inhibits E. coli growth and survival by suppressing all the subunits of RNAP core enzymes (Fig. 4A and Table 1). This distinctive mechanism of action suggests that RJ could offer an alternative approach to combat bacterial resistance, making it a promising candidate for natural antibacterial strategies. Further research and clinical studies are necessary to fully understand the potential of RJ as an effective and sustainable solution against bacterial resistance.
In addition to its ability to inhibit transcription, RJ also exerts an influence on the translational process mediated by ribosomal proteins, specifically targeting the ribosome (Fig. 4B). Antibiotics, which are broadly classified as agents that hinder protein synthesis, can be categorized into two primary subclasses based on their binding sites on ribosome: the 50S inhibitors and 30S inhibitors 6. In this study, we found that the treatment of 20 mg/mL RJ resulted in the down-regulation of 44 ribosomal proteins, encompassing 80% of 30S subunits and 77% of 50S subunits (Table 2). These findings shed light on the central role of RJ in regulating protein synthesis and maintaining translation fidelity (Fig. 4B). Intriguingly, RJ suppressed ribosomal proteins linked to "response to antibiotic", including RplF, RplV, RplD, RpsE, RpsQ, RpsD and RpsL (Fig. 4B), suggesting potential benefits in combating resistance and enhancing treatment efficacy.
ROS which include not only O2•− but also other oxygen-derived molecules like H2O2 and hydroxyl radicals (•OH), form during metabolic processes and exposure to stressors like low temperatures, chemicals, and UV light21. Improper ROS regulation, due to their high reactivity, can cause oxidative stress, resulting in cellular damage and dysfunction, including DNA mutations, protein misfolding, and lipid peroxidation, etc 22. Bacteria employ an antioxidant defense system that includes enzymes like SOD and catalase, to mitigate the harmful effects of ROS. In the case of E. coli, it possesses two cytoplasmic SOD enzymes (MnSOD encoded by sodA, and FeSOD encoded by sodB), and two catalases known as HPI and HPII (encoded by katG and katE, respectively).
Antibiotics lethality is often accompanied by ROS generation 8,23. In our current investigation, it has been uncovered that the natural product RJ significantly elevates levels of ROS and MDA, established markers of bacterial oxidation (Fig. 5C, D)24. This increase in oxidative stress correlates with dysfunction of key antioxidant enzymes, such as SodA, SodB, and KatG, as illustrated in Figs. 1E, 5A, and 5B. However, the expression level of katE did not show significant changes between the RJ-treated group and the control group (as indicated by RT-qPCR results and proteomic analysis, data not shown), suggesting that this gene is not strongly associated with the antibacterial effects of RJ. Our results suggest that RJ-induced ROS accumulation in E. coli was resulted from the inhibition of the antioxidant enzymatic system, ultimately leading to cellular damage.
The most remarkable discovery in this study is the identification of host TFs responsive to RJ treatment (Fig. 6A, 6B). CRP, a central regulator on top of the hierarchical regulatory framework, playing a vital role in global stress responses such as oxidative stress, low pH, and osmotic pressure25. CRP alterations or amino acid substitutions impact hundreds of genes26. Particularly, it has been verified that E. coli crp mutant exhibited strong resistance to oxidative stress27. In this study, RJ strongly inhibited the expression of crp even with low-dose treatment (Fig. 6B). As a result, the control exerted by CRP over the TCA cycle and cellular responses to oxidative stress was substantial (Fig. 6A; Table S3, S4). The TCA cycle serves as the ultimate converging route for the oxidative breakdown of fuel molecules (amino acids, fatty acids, and carbohydrates) in E. coli.
Following RJ treatment, the key enzymes in the TCA cycle under the control of CRP exhibited downregulation, including mdh, sucB, sucC, sucD, and gltA (Table S2). As an effect of RJ treatment, the loss of the certain enzymes of TCA cycle resulted from CRP downregulation, may lead to reduced pool of NADH and decreased production of superoxides, as indicated by Kohanski et al.8. In addition, pentose phosphate pathway (PPP), a route to stabilize NADPH levels that used as cofactor to reduce ROS through antioxidant systems28, was indirectly regulated by CRP through the downstream TF gene dksA and the downstream hub proteins TalA and TktB (Table S3, Fig. 6A). Our results indicated that RJ treatment disrupts energy production and electron carrier production from the TCA cycle by repressing CRP, while reduced CRP expression may serve as a bacterial strategy to counteract RJ-induced oxidative damage.
IHF is a global regulator operating in genetic recombination and exerts control over transcriptional and translational processes in gram negative bacteria29. IHF-DNA binding is influenced by environmental factors like ion concentration30, though the precise role of environmental conditions in regulating its function remains unclear. In the present study, we report IHF's negative response to diverse RJ concentrations (Fig. 6B, Table S2), disrupting bacterial growth by affecting multiple cellular processes, especially the TCA cycle (Fig. 6A). Recent research has unveiled IHF's role in regulating drug tolerance at low IHF levels, particularly its ability to repress the transcription of isocitrate dehydrogenase (Icd), a key component of the TCA cycle, while simultaneously activating the expression of isocitrate lyase (AceA), the first enzymes in the glyoxylate bypass31. We identified a number of TCA cycle genes and glyoxylate bypass genes controlled by IHF were significantly repressed by RJ, including citrate synthase (GltA), Icd, 2-oxoglutarate dehydrogenase E2 subunit (SucB), succinyl-CoA synthetase subunit α and β (SucD and SucC), malate dehydrogenase (Mdh), AceA and malate synthase A (AceB) (Table S2). Together with the recent results on the role of CRP, DksA and IHF play in bacterial antibiotic tolerance and persister formation31–33, this study elucidated the important roles of CRP, DksA and IHF in response to RJ treatment, and emphasize the intricate connections among the biological process modules that governed by the key TFs. The observed changes in Crp, DksA and IHF in RJ-treated samples indicate their critical importance for bacterial survival under the stress imposed by RJ.