Mechanism of geraniol against E. coli infection by regulating YDIV

doi:10.21203/rs.3.rs-3869163/v1

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Mechanism of geraniol against E. coli infection by regulating YDIV

https://doi.org/10.21203/rs.3.rs-3869163/v1

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Purpose

Geraniol, an active component found in the essential oil of various medicinal plants, possesses a wide range of antibacterial properties, including against E. coli. Nevertheless, the precise inhibitory mechanism of geraniol on E. coli remains elusive.

Methods

Co-cultivation of multidrug-resistant Escherichia coli with geraniol was performed to investigate changes in biomass, gene expression, intracellular iron concentration, phagocytic activity, and complement-mediated killing ability. Additionally, protein expression alterations were assessed to evaluate the regulatory effects of geraniol.

Results

The compound geraniol demonstrated a downregulation of ydiv and iron metabolism gene fepA, fecB and fhuF expression. Concomitantly, it was observed that linalool led to a decrease in intracellular iron ion concentration. Meanwhile, treatment of E. coli with geraniol resulted in a downregulation of ompW gene expression. Additionally, it led to a decrease in the killing ability of antiserum complement and an increase in the phagocytic capacity of macrophages. Furthermore, geraniol demonstrated augmented binding effects, possibly facilitated through hydrogen bonding, relying on structural simulation or MST.

Conclusion

These results suggested for the first time that geraniol by regulating the iron transport-related proteins YdiV, thereby decreasing the ability of antiserum complement, and an increase in the phagocytic capacity of macrophages exerting inhibited E. coli.

Biological sciences/Microbiology/Bacteria/Infectious disease epidemiology

Biological sciences/Microbiology/Antimicrobials

Escherichia coli

antimicrobial resistance

geraniol

iron

Gram-negative bacteria (GNB) exhibit high morbidity and mortality rates in infections due to their resistance to antibacterial drugs, posing a significant global health risk. Of utmost concern is the alarming proliferation of carbapenem-resistant gram-negative bacteria (CR-GNB), which poses a grave public health threat. Despite the introduction of various antimicrobial combinations to enhance their efficacy against bacterial infections, there remains a persistent clinical need for more potent antibiotics [1–3]. Natural products dominate the preferred chemical scaffolds for the discovery of antibacterial agents [4]. Traditional Chinese medicines have provided natural and unique advantages in the treatment of infectious diseases. Therefore, it is important to further develop and utilize them for the treatment of clinical infections caused by drug-resistant bacteria [5]. Due to its notable antimicrobial activity, geraniol emerges as a promising candidate for potential antibacterial therapy [6, 7]. Iron is an indispensable elemental component across all life domains, serving as a vital cofactor in a multitude of enzymes crucial for metabolism and growth [8]. The presence of excessively high or abnormally low iron concentrations exerts detrimental effects on both growth and metabolic processes [9]. Therefore, it is important to maintain iron at a reasonable level within the cell. YdiV-SlyD-Fur is a crucial pathway for regulating iron ions in E. coli, with YdiV serving as the primary regulator [10]. Studies have shown that the ydiv gene is induced under low-iron conditions, but this induction is not related to Fur [11]. YidV and SlyD play a crucial role in facessential for the survival and growth of E. coli, aiding in iron ion absorption and evasion of the host's innate immune system to promote bacteria survive [12]. Therefore, we hypothesize that geraniol disrupts intracellular iron uptake in E. coli by regulating YdiV. This could serve as a new target for the development of novel antimicrobial drugs that inhibit E. coli.

The growth-affecting capabilities of geraniol against MDR 25922

After the MDR 25922 was cultured in microwells containing different concentrations of geraniol for 24-hour, the MIC value was measured as 686 µg/mL. The growth of MDR 25922 in MHB medium containing different concentrations of geraniol was detected by microplate reader (Fig. 1), compared with the control group, different concentrations of geraniol had an influenced the growth of MDR 25922, of which 1/2 MIC had the most obvious effect, prolonging the time to enter the logarithmic growth phase and significantly reducing the bacterial amount in the plateau phase. Geraniol had good in vitro direct antibacterial activity.

Geraniol regulated down regulation of iron metabolism related genes

After incubation in geraniol-containing MHB medium for 4-hour, bacteria were collected by centrifugation, RNA was extracted after lysis, and reverse transcription was performed. Quantitative real-time PCR was utilized to evaluate alterations in gene transcriptional levels. The results showed that the transcription level of ydiv, fepA, fecB, fhuF, ompW was significantly decreased in intracellular bacteria compared to bacteria grown under normal conditions (Fig. 2).

Geraniol induces a reduction in intracellular iron content

To elucidate the impact of geraniol on iron metabolism, we conducted an assessment of intracellular iron content. The results indicated a dose-dependent reduction in intracellular iron content following treatment with geraniol at concentrations of 1/8 and 1/4MIC (Fig. 3).

Geraniol promoted the ability of RAW264.7 cells to phagocytose

Initially, cellular safety evaluation of geraniol was performed. The results revealed a negative correlation between the viability of RAW264.7 cells and the concentration of geraniol within the range of 1/2 MIC to 1/16 MIC. Specifically, no significant difference was observed in the impact on cell viability between the control group and geraniol at 1/16 MIC and 1/8 MIC concentrations (Fig. 4a), which could be used for subsequent experiments. The results showed that geraniol of 1/16 MIC and 1/8 MIC promoted the ability of RAW264.7 to engulf NR and had a significant dose-response relationship (Fig. 4b).

Geraniol reduced the ability of MDR 25922 to counter serum complement killing

Using flow cytometry-based sorting, according to the different fluorescence intensities, the bacteria were sorted in different regions (Q2-1 was the dead bacteria and Q2-2 was the live bacteria). It was observed that even in the presence of 1/4 MIC of geraniol, 92.28% of bacterial cells remained viable. However, upon escalating the concentration of geraniol to 1/2 MIC, the proportion of viable bacterial cells decreased to 79.56%. The findings demonstrated that geraniol has the ability to attenuate the serum complement resistance of MDR 25922 (Fig. 5).

Binding of geraniol to YdiV protein

Analyzed of the Binding of geraniol and YdiV Protein by Using Molecular Docking and MST. The molecular docking result showed that there were hydrogen bonds between geraniol and YdiV protein (Fig. 6). The MST result showed that the dissociation constant KD value of geraniol and YdiV protein was 2.1066E-05M (Fig. 7), indicating that geraniol and YdiV had an excellent binding effect.

Figure 6 The results of geraniol docking with MDR 25922 YdiV protein and showed the formation of hydrogen bonds with ARG-19, ARG-237, GLN-23 of YdiV protein.

The escalating bacterial resistance to antimicrobial agents represents a critical global issue demanding immediate attention [13]. E. coli has emerged as a prominent contributor to antimicrobial resistance, with MDR strains becoming increasingly prevalent [14]... This is particularly worrying given the current paucity of new compounds active against MDR-GNB [15]. Traditional herbal medicine has a long-standing history of human utilization in China, where it is documented and extensively covered in dedicated monographs within the Chinese Pharmacopoeia. There is accumulating evidence demonstrating the antimicrobial properties of a growing number of herbs including garlic, Tsaoko-Fructus, black cumin, cloves, cinnamon, thyme, allspice, bay leaves, mustard, and so on [16]. This has prompted an interest in herbal therapy for the treatment of bacterial infections disease. The herbs and their associated bioactive ingredients that can potentially enhance the effects of current antibiotics [17]. The extensive history of their application attests to their high clinical safety, making the screening of potential antibiotic alternatives from them a potentially shorter development process compared to developing a novel antibiotic [18]. Geraniol is a terpenoid compound present in a variety of Chinese herbal medicines. It has antibacterial, anti-inflammatory, antioxidant, and other pharmacological activities, and most of these have been tested in vitro [12, 19–21]. However, its medicinal mechanism is unclear. This study demonstrates the inhibitory activity of geraniol against MDR E. coli, along with its ability to delay entry into the logarithmic growth phase and reduce biomass.

Iron serves as an indispensable micronutrient for bacteria, participating in a multitude of vital biological pathways, including DNA synthesis and energy metabolism. Furthermore, it plays a pivotal role in determining bacterial virulence [22]. In the host, iron usually binds tightly with biomolecules like heme, causing an iron-depleted environment in vivo, in which bacteria have to adapt to by employing a series of iron acquisition mechanisms [23]. One of the strategies is siderophore, which has a high affinity for iron ions that can capture iron from the host’s protein iron complexes. Another way for uptake of iron relies on direct contact between the pathogen and the iron source [24–26]. In the preliminary investigation, a downregulation of iron metabolism-related pathways was observed through transcriptomic analysis. Therefore, the expression levels of relevant genes in Escherichia coli were initially validated following co-cultivation with geraniol. Subsequent analyses confirmed a reduction in intracellular iron content. In numerous investigations, we have observed a consistent phenomenon wherein the inhibition of bacterial growth is achieved through the suppression of iron ion metabolism in various pathogens, such as Vibrio cholerae, Pseudomonas aeruginosa, and Klebsiella pneumoniae [27, 28]. Various regulatory systems play crucial roles in iron metabolism. The global regulatory protein CsrA has been implicated in the uptake and storage of iron in E. coli, underscoring its significant role in modulating iron homeostasis [29]. Meanwhile, YdiV exerts a pivotal role in the iron uptake pathway of Escherichia coli by manipulating the DNA binding ability of Fur via a SlyD-dependent mechanism. Hence, YdiV is critical for regulating iron acquisition in this microorganism [10]. The downregulation of ydiv indicates that geraniol suppresses the iron metabolism pathway in E. coli. Simultaneously, other iron metabolism genes (fepA, fecB, fhuF, ompW) associated with the regulatory factor ydiv exhibit a downregulation trend. Collectively, these investigations have underscored the critical role of iron ion concentration in bacterial growth, thus highlighting the potential of regulating iron metabolism as a novel target for antibacterial drug development. According to the results, it is speculated that geraniol can bind to YdiV, causing abnormal iron ion metabolism of bacteria and reducing the concentration of intracellular iron ions.

As a component of the host's innate immune system, macrophages play a crucial role in bacterial infections by phagocytizing and eliminating invading pathogens [30]. Studies have demonstrated that the natural compound resveratrol exerts anti-inflammatory effects by modulating anti-inflammatory cytokines and immune responses [31]. Meanwhile, geraniol exhibits the ability to reduce inflammatory cytokines and attenuate tissue infiltration of neutrophils in mice infected with methicillin-resistant Staphylococcus aureus (MRSA) [7]. Citral, which is also a vital constituent of traditional herbal volatile essential oils, exhibits notable anti-MRSA activity in vivo, possibly by attenuating inflammatory responses and combating oxidative stress to confer protective effects on the host organism [32]. The ability of eugenol to geraniol the phagocytic capacity and complement-mediated killing activity of neutrophils may parallel the mechanism by which certain natural compounds augment immune function and exert anti-inflammatory effects. It is speculated that geraniol may also enhance the host immunity, which requires verification through further in vivo experiments.

This study provides a comprehensive understanding of geraniol, which will be beneficial for exploring the mechanism and potential medicinal applications of it, as well as developing a rational quality control system for geraniol as a medicinal material in the future. Meanwhile we will provide high-quality evidence to help patients, clinicians as well as health policymakers select better treatment strategy of bacterial infection disease.

Cell lines, bacterial strains

RAW264.7 cells were obtained from the Procell Life Science&Technology Andre and cultured in special media (Procell, WuHan, China) supplemented with 10% fetal bovine serum. MDR 25922 was the ATCC 25922 induced by amoxicillin showed obvious cross-resistance to cephalosporins, piperacillin and ampicillin/sulbactam antibiotics commonly used in clinical practice, and produced ESBL, but remained sensitive to the geraniol, which were from our own laboratory collection. The bacteria were grown at 37 ℃ grown in Luria Broth (LB) medium or Muellere Hinton Broth (MHB) medium (Solarbio, Beijing, China).

Minimum Inhibitory concentration (MIC) determination

The MIC value of geraniol (Sigma-Aldrich, USA, 1003223610, 98%) against MDR 25922 was determined by microbroth dilution. Briefly, added 100 µL of each of MHB medium was added to the 96-well plate and geraniol was diluted by double dilution through 12 concentration gradients. Then added 100 µL of diluted bacterial solution to each well, the final concentration of the drug was 0.021–43.02 mg/mL, and the MHB culture base was used as the blank control, the bacterial solution without the drug was used as the positive control, and the drug without the bacterial solution as the negative control. After incubation in a 37°C incubator for 18–24 hours, the lowest drug concentration with no bacterial growth was the MIC.

Growth curve of the MDR E. coli under the geraniol

After activation of MDR 25922, individual colonies were plated in normal saline and adjusted to 0.5 Mc. An equal amount of each bacterial culture was then transferred to the MHB containing geraniol at a ratio of 1:200 (v/v) and incubated at 37℃ at 200rpm. The OD₆₀₀ was monitored at 2-hour intervals using a spectrophotometer (BMG Labtech, GER). The experiment was repeated three times, and all samples were measured in triplicate.

Quantitative reverse transcription PCR (qRT-PCR) analyses

Total RNA was extracted from MDR 25922 and treated with geraniol using an RNA extraction kit (AG, Hunan, China). The RNA was then converted to cDNA (Vazyme, Nanjing, China) using reverse transcription. qRT-PCR was performed using SYBR Green Master Mix (AG, Hunan, China) to detect transcript levels of ydiv, fepA, fecB, fhuF and ompW were detected, with 16S serving as the housekeeping gene. The fold change was calculated using the 2^−ΔΔCt Method. The primer sequences are shown in Table 1.

Table 1 The primer sequences

Intracellular iron concentration detection

Geraniol and MDR 25922 were added to MHB culture medium and cultured at 37°C with shaking at 200 rpm. The bacteria were collected, and the intracellular iron concentration was detected using colorimetry (Applygen Technologies Inc). The absorbance 550 nm was determined, a standard curve was plotted, and the iron concentration was calculated.

Neutral red phagocytosis experiment

Effects of the geraniol on cell viability. The cell concentration was adjusted to approximately 5×10⁴ cells/mL and 100µL was seeded per well in a 96-well plate. The plate was then incubated at 37°C in a 5% CO₂ incubator for 12-hour. After discarding the culture medium, a special medium containing different sub-inhibitory concentrations of geraniol was added. A normal control group and blank medium control group were set up, after 4-hour of culture, 10µL of CCK8 was added per well and incubated for 2-hour. The OD value was determined at a wavelength of 450nm using a microplate reader.

Neutral red phagocytosis experiment. The geraniol concentration that no effect on cell viability was screened from the CCK8 results. Set the cell concentration to about 8 ×10⁵ pcs/mL, 100µL per well. Incubated in the cell culture incubator for 12h and washed the culture 2 times, then added geraniol containing medium to treat the cells for 4h. Discarded the supernatant and added 100µL of 0.04% neutral red solution, cultured in the cell culture incubator for 2h, washed 2 times with PBS and made it to dry. Finally, added 200µL of lysate (ethanol: acetic acid 1:1) per well and put it in a shaker at room temperature for 10 min, OD values were measured at a wavelength of 540 nm with a microplate reader.

Effect of geraniol on serum complement killing MDR 25922

Fresh and heat-inactivated guinea pig serum were collected (incubated at 56°C for 30 min). The bacteria were collected after shaking at 37°C for 4-hour. MDR 25922 was inoculated in MHB culture medium with varying concentrations of geraniol and fresh serum (100µL culture added to 5µL serum), respectively. Negative controls included no serum and heat-inactivated serum, while isopropanol treatment was used as a positive control. The study aimed to analyze the effect of geraniol on MDR 25922 resistance to serum complement killing. After staining with the Live Bacteria/Dead Bacteria Double Staining Kit (Thermo, NYC, USA), the survival rate of bacteria in each group was detected by flow cytometry (Selected the PI/SYTO dual staining fluorescence channel procedure and aspirate 5000 bacteria for analysis.)

Geraniol interaction with the regulatory protein YdiV protein

The YdiV protein was obtained from the PDB library. Small molecules and proteins were added using Schrodinger. Geraniol small molecules were prepared in the Lipper plate. The protein preparation module was optimized to prepare the protein, and the binding site was defined with the sitemap. The docking box was generated, and the XP molecule docking was carried out to obtain the preliminary binding effect.

Microscale thermophoresis (MST) analysis of geraniol binding to YdiV

RED-NHS second-generation labelling kit (Mabnus, Wuhan, China), RED-NHS second-generation dye labelled the purified YdiV protein, after the pre-test and binding check detection were completed, geraniol was dissolved in DMSO and diluted to 1mM, diluted to 16 tubes by 2-fold concentration gradient, each tube solution was 10µL after dilution, 10µL of diluted protein solution was added to mix, and the capillary was pipette red and placed in MST instrument for detection.

Statistical analysis

Data were expressed as mean ± SD, with statistics carried out in the GraphPad Prism 5. Results were analyzed using ordinary one-way ANOVE Multiple comparisons to compare the mean of each column with the mean of a control column. p < 0.01 is extremely significant, p < 0.05 is significant.

Author contributions: W.G. and M.D. were responsible for conceptual design, Z.P. and M.D. provided financial support, N.L. provided technical support, Y.W. prepared Figures 1-4 and tables, while Y.Z. prepared Figures 5 and 6. Y.W., Y.Z., and N.L. wrote the main manuscript text. N.L. writing-review & editing. All authors reviewed the manuscript.

Declaration of interest: None.

Funding: This study was supported by National Natural Science Foundation of China (31970137, 82204601), the special project of Liyan workshop aesthetic medicine research center of Chengdu medical college (21YM007), the Sichuan Science and Technology Program (2020YJ0401), Project funded by China Postdoctoral Science Foundation (2021M703134) and Sichuan Provincial Administration of Traditional Chinese Medicine Innovation Team Project (2023ZD02).

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No competing interests reported.

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You are reading this latest preprint version

Mechanism of geraniol against E. coli infection by regulating YDIV

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Results

The growth-affecting capabilities of geraniol against MDR 25922

Geraniol regulated down regulation of iron metabolism related genes

Geraniol induces a reduction in intracellular iron content

Geraniol promoted the ability of RAW264.7 cells to phagocytose

Geraniol reduced the ability of MDR 25922 to counter serum complement killing

Discussion

Materials and methods

Cell lines, bacterial strains

Minimum Inhibitory concentration (MIC) determination

Quantitative reverse transcription PCR (qRT-PCR) analyses

Intracellular iron concentration detection

Neutral red phagocytosis experiment

Effect of geraniol on serum complement killing MDR 25922

Geraniol interaction with the regulatory protein YdiV protein

Microscale thermophoresis (MST) analysis of geraniol binding to YdiV

Statistical analysis

Declarations

References

Additional Declarations

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