Repurposing Rifaximin against Klebsiella pneumoniae via Targeting of Transcription Anti-termination Protein RfaH for Novel Antimicrobial Development

Repurposing Rifaximin against Klebsiella pneumoniae via Targeting of Transcription Anti-termination Protein RfaH for Novel Antimicrobial Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Repurposing Rifaximin against Klebsiella pneumoniae via Targeting of Transcription Anti-termination Protein RfaH for Novel Antimicrobial Development Anam Ashraf, Arunabh Choudhary, Mohammad Ali Khan, Saba Noor, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4724428/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anti-termination protein RfaH plays a crucial role in promoting virulence across various Gram-negative pathogens, including Klebsiella pneumoniae (KP). RfaH directly interacts with RNA-polymerase and ribosomes, which in turn facilitates the activation of operons associated with capsule, cell wall, and pilus biosynthesis. This study aimed to investigate the repurposing potential of rifaximin, a well-established antibiotic, against KP by strategically targeting RfaH, a pivotal anti-terminator protein in transcription. Fluorescence studies observed an excellent binding affinity between rifaximin and RfaH ( K a = 7.38 x 10 6 M −1 ). Intriguingly, rifaximin treatment causes a significant reduction in capsule production in KP when compared to untreated controls, elucidating its inhibitory influence on RfaH activity. The minimum inhibitory concentration for Rifaximin was calculated as 100µM and a minimum bactericidal concentration of 200µM against KP (ATCC 700603 strain). Docking and MD simulation studies provided detailed atomic insights into the Rifaximin binding to RfaH and structural dynamics of the RfaH-Rifaximin complex. These multifaceted findings collectively investigated the potential of rifaximin as a repurposed antibiotic against KP. Finally, a strong interaction of RfaH with rifaximin and subsequent inhibition of the growth of KP provides a novel avenue for antimicrobial development for addressing the persistent global challenge of antibiotic-resistant infections. Rifaximin Anti-termination Protein RfaH MD simulation Fluorescence studies Anti-biotic resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Klebsiella pneumoniae (KP), a ubiquitous Gram-negative bacterium, is a significant cause of community and healthcare-associated infections [ 1 ]. Multidrug-resistant Klebsiella pneumoniae (MDR-KP) has emerged as a formidable threat to global health, posing a significant challenge in the field of infectious diseases. This bacterium is notorious for its ability to evade the effects of a wide range of antibiotics, rendering treatment options scarce and compromising patient outcomes [ 2 ]. The rising prevalence of antimicrobial resistance (AMR) in KP poses a substantial problem to healthcare systems worldwide due to its alarming resistance to multiple classes of antibiotics, including aminoglycosides, fluoroquinolones, cephalosporins, and even carbapenems, considered the last-line of defense against bacterial infections [ 3 ]. The alarming rise of multidrug-resistant (MDR) and hypervirulent (hv) Klebsiella pneumoniae (MDR-hvKp) convergent clones necessitates active surveillance networks and robust monitoring systems to track and manage the spread of these highly successful hybrid pathogens [ 4 ]. Drug repurposing is a powerful and rapidly evolving strategy that offers a promising avenue for accelerating drug discovery and development [ 5 ]. It aims to address the lengthy and resource-intensive nature of conventional drug development. By focusing on rationally understanding and redefining the therapeutic potential of existing drugs, we can leverage the established safety profiles and bypass the extensive clinical trial phases associated with new drug development. This approach resonates with the growing trend in drug discovery, where computational methods are increasingly employed to rapidly identify promising targets for either the repurposing of existing drugs or the rational design of novel chemical entities, accelerating the fight against emerging infectious diseases [ 6 , 7 ]. Its numerous advantages, including reduced cost, faster development times, and established safety profiles can be particularly beneficial in addressing multidrug-resistant infections or targeting complex diseases characterized by multiple underlying mechanisms [ 8 , 9 ]. Rifaximin is an orally administered antibiotic with minimal systemic absorption. It possesses broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria [ 10 ]. Rifaximin, a non-aminoglycoside antibiotic is a semisynthetic derivative of the natural antibiotic Rifamycin. It is a structural analog of rifampin, another antibiotic derived from Rifamycin [ 11 ]. The irreversible nature of Rifaximin binding to RNA polymerase ensures sustained inhibition of bacterial RNA synthesis, providing a prolonged therapeutic effect. This characteristic contributes to the effectiveness of rifaximin in treating various bacterial infections [ 12 ]. RfaH, essential for virulence in pathogens like E. coli and K. pneumoniae , activates virulence genes through intricate interactions with RNA polymerase and ribosomes [ 13 , 14 ]. This dual action promotes transcription and translation of key operons involved in capsule, cell wall, and pilus biosynthesis, ultimately enhancing bacterial virulence [ 15 ]. In the past decades, researchers have uncovered a group of operons whose full function relies on the protein RfaH in various bacterial strains. These operons are responsible for producing vital components of bacteria, including the protective LPS core [ 16 ], the O-antigen on the surface [ 17 ], the α-hemolysin toxin [ 18 ], the F-factor for genetic exchange [ 19 ], the hemin receptor for iron uptake [ 20 ], and even the group I, II, and III capsules that shield the bacteria [ 21 ]. When RfaH is absent, the expression of these operons diminishes, leading to a significant decrease in the levels of these vital components, ultimately affecting bacterial viability, virulence, and interaction with the host immune system [ 22 ]. RfaH acts as an anti-terminator, counteracting the effect of terminators and allowing for the complete transcription of operons. Understanding RfaH's role in suppressing operon polarity provides valuable insights into gene regulation in bacteria [ 23 ]. This, in turn, targeting RfaH can have a significant impact on bacterial health and virulence, affecting their ability to withstand environmental stress, evade the immune system, and cause disease [ 24 , 25 ]. Given the critical role of RfaH in KP survival and virulence, targeting this protein with repurposed drugs presents an attractive therapeutic strategy [ 14 , 26 ]. Rifaximin, a known antibacterial agent, emerged as a promising candidate in this study. Utilizing in vitro assays and spectroscopic techniques, we established the inhibitory effect of Rifaximin on KP, likely through its interaction with RfaH. Furthermore, molecular dynamics (MD) simulations corroborated our experimental findings, lending additional support to the potential of rifaximin as a repurposed therapeutic option for KP infections. These results warrant further investigation of the efficacy of rifaximin and the mechanism of action against KP, potentially leading to the development of a novel therapeutic approach for combating this critical pathogen. Methods Cloning, Expression and Purification of RfaH The RfaH gene of Klebseilla pneumoniae was cloned commercially using gene-specific primers with 6X His tag (GenScript Biotech, NJ, USA). The protein was expressed in E. coli strain BL21 expression cells and purified using the Ni-NTA column. Briefly, the construct of RfaH was PCR-amplified and cloned into the pET28b(+) vector. Restriction digestion and DNA sequencing verified the resulting clone. For maximum expression, RfaH was induced with 0.5 mM IPTG and incubated for 16 hours at 16°C post-induction. The cells were then lysed using a sonicator in lysis buffer (Tris 20mM NaCl 200mM PMSF 7.5mM and DTT 0.1mM). The resulting lysate was loaded on the Ni-NTA column and purified using an imidazole concentration gradient. The purified RfaH was eluted and analyzed on SDS-PAGE and confirmed by mass spectrometry. Fluorescence measurements A fluorescence quenching experiment was performed on a Jasco spectrofluorometer (FP 8200, Japan) to assess the interaction of rifaximin with RfaH. Rifaximin was first dissolved in DMSO to generate a 50 mM stock solution, which was subsequently diluted to a working concentration of 0.5 mM using a Tris-NaCl buffer. With a fixed concentration of RfaH at 8 µM, rifaximin was titrated in a 1:9 ratio until saturation. Emission spectra were recorded within the 300–400 nm range upon excitation of tryptophan at 280 nm. Slit widths and sensitivity settings were standardized for all measurements. The obtained data was analyzed using the modified Stern-Volmer equation to determine the binding affinity ( K a) and number of binding sites ( n ) for rifaximin on RfaH. Triplicate measurements were conducted for each experiment, with appropriate corrections applied for buffer blanks and inner filter effects. This comprehensive approach ensured an accurate and reliable evaluation of the interaction between rifaximin and RfaH using our well-established protocol [ 27 – 29 ]. Minimum inhibitory concentration measurements The minimum inhibitory concentration (MIC) of rifaximin against K. pneumoniae (ATCC 7006043) using the INT (p-iodonitrotetrazolium chloride) colorimetric assay was performed. Rifaximin was dissolved in DMSO, ensuring its concentration remained below 2.5% to avoid affecting bacterial growth. The test solution was serially diluted in broth media in a 96-well plate, followed by the addition of 100 µL inoculum containing 1.5 x 10 5 CFU/mL of bacteria per well. The plate was sealed, shaken, and incubated at 37°C for 18 hours. Negative controls included KP cultures without any inhibitor and media alone. A positive control involved KP cultured with ciprofloxacin, a known antibiotic. The assay was repeated three times for accuracy. After incubation, 0.2 mg/mL INT was added to each well, and the plate was further incubated for 30 minutes. Viable bacteria reduced the yellow dye to pink, indicating their presence. The MIC was defined as the lowest concentration of rifaximin that prevented color change and completely inhibited bacterial growth [ 30 ]. Determination of minimum bactericidal concentration Time-killing curves were employed to explore the bactericidal effects of Rifaximin against K. pneumoniae (ATCC 700634). Freshly prepared bacteria in their early logarithmic growth phase were exposed to Rifaximin at various concentrations, ranging from 0.5 to 5 times the MIC. This range aimed to capture both potential killing and stalling effects, as the MIC might only halt growth without reducing the initial bacterial count. Controls included bacteria without inhibitors (growth control) and media alone (sterility control). At specific time points (3, 6, 9, 12, and 24 hours), samples were taken, diluted, plated, and incubated for 24 hours to count the remaining colony-forming units (CFUs). The CFU data was then plotted on a logarithmic scale (y-axis) against time (x-axis) to visualize the killing activity of Rifaximin over time. Each experiment was repeated three times for statistical accuracy. This setup allowed researchers to determine the time-dependent killing effectiveness of Rifaximin against KP [ 31 ]. Capsule quantification assay and visualization To investigate the impact of Rifaximin on KP capsular polysaccharide (CPS) production and uronic acid content we did a capsule quantification assay explained elsewhere [ 32 , 33 ]. Briefly, 20 ml LB broth cultures with and without 100µM Rifaximin were inoculated with K. pneumoniae (ATCC 700603) and incubated for 16 hours. Cell density was normalized by CFU/ml determination. A modified Zwittergent extraction protocol was employed as follows [ 15 ], 500 µL cultures were incubated with 1% Zwittergent in 100mM citric acid at 50°C for 20 min and then centrifuged at 10,000g for 5 min. The supernatants were precipitated with cold ethanol by incubating it at 4°C for 20 min. After centrifugation at 10,000g for 5 min the pellets were resuspended with water and treated with 12.5 mM sodium tetraborate in concentrated sulfuric acid. The samples were then boiled at 95°C for 5 min and reacted with 0.15% 3-phenylphenol in 0.5% NaOH. Absorbance at 520nm was measured, normalized to CFU/ml, and averaged from triplicate assays, providing a quantitative measure of uronic acid content relative to bacterial density. This approach allowed a comparison of CPS production and uronic acid content in KP with and without Rifaximin treatment [ 34 ]. The outer capsule of the cells was visualized by negative staining using log phase K. pneumoniae (ATCC 700603) culture, which was mixed with 7% nigrosin dye. Rifamixin-treated and untreated cells were visualized under the microscope. Molecular docking To unveil the interactions between the KP RfaH protein and the antibiotic Rifaximin molecular docking was done. The 3D structure of RfaH was modeled from AlphaFold (ID: AF-W1HRW8-F1). Subsequently, the structure was refined through refinement processes utilizing Swiss PDB Viewer and MGL tools. MGL tools added hydrogen atoms and assigned Kollman charges to RfaH's polar groups, mimicking its real-life chemistry. InstaDock [ 35 ] was used to virtually dock Rifaximin onto RfaH, exploring numerous binding possibilities like an exhaustive eight-round search. Finally, Discovery Studio Visualizer [ 36 ] and PyMOL [ 37 ] were used to analyze the resulting protein-ligand complexes, revealing the intricate interactions between RfaH and Rifaximin. MD simulations MD simulation stands as a crucial in-silico technique for understanding the structural dynamics and interactions between a ligand and a protein molecule [ 38 ]. The stability and structural dynamics of the RfaH protein bound to Rifaximin were assessed through a simulation lasting 100 ns. The simulation of both the RfaH-Rifaximin complex and the native RfaH protein was conducted using GROMACS version 5.5.1 [ 39 ]. Two configurations were established for the protein and protein-ligand complex. In each configuration, a cubic box was generated, maintaining a 10Å distance from the protein and incorporating the simple point-charge (SPC16) water model. To eliminate potential steric hindrance among atoms, an energy minimization technique was applied to the solvated systems. This involved 1500 steps of the steepest descent method for energy minimization. A two-step equilibration lasting 100 ps, incorporating periodic boundary conditions, was subsequently conducted. Following this, a simulation of 100 nanoseconds was executed for each configuration, and the resultant trajectories were analyzed utilizing the 'gmx' tools [ 39 ]. Results The alarming rise of antimicrobial resistance (AMR) in KP necessitates a multi-pronged approach, including judicious antibiotic use, enhanced infection control, and continual exploration of novel therapeutic strategies [ 40 ]. Repurposing existing drugs, for example, the rapid FDA approval of Remdesivir for COVID-19 [ 41 ], presents a promising avenue for combating KP infections. Our study lays the groundwork for repurposing Rifaximin against KP infections. While its poor gut absorption may pose challenges in in-vivo studies our in-vitro data demonstrate its antibacterial activity against KP. This paves the way for designing novel RfaH inhibitors based on rifaximin scaffold, potentially with improved gut absorption. By synergistically integrating insights from disease pathophysiology, drug pharmacology, and computational analysis, we can prioritize promising drug candidates for KP infections. This interdisciplinary approach holds immense potential for accelerating the development of effective KP therapies, ultimately improving patient outcomes and mitigating the healthcare burden. Cloning, expression and purification of RfaH The RfaH gene was cloned into pET-28a(+) vector using gene-specific primers with 6X His tag. The positive colonies were confirmed by using restriction digestion enzymes Nde I and Xho I ( Fig. 1 A ). The plasmid was then transformed into DH5α to increase the copy number and in BL21 (DE3) cells for expression. The positive clones were screened using a specific antibiotic kanamycin, which was further confirmed by sequencing ( Fig. 1 B ). His-tagged recombinant RfaH protein was purified using Ni-NTA affinity chromatography. Eluted fractions were analyzed by SDS-PAGE depicting a single purified band ( Fig. 1 C ). Fluorescence binding studies To investigate the antibacterial effect of Rifaximin through RfaH inhibition, we employed fluorescence binding studies. The aromatic residues, particularly tryptophan, tyrosine, and phenylalanine, have unique fluorescence properties that are influenced by factors like solvent polarity, temperature, and interactions with other molecules. When a ligand binds to a protein, it can alter the local environment around these aromatic residues, leading to changes in their fluorescence emission intensity, spectra, or lifetime. This helps to monitor the formation and dynamics of protein-ligand complexes in real-time, providing valuable insights into binding affinity, specificity, and potential mechanisms of action. We used a quenching approach to assess the interaction between Rifaximin and RfaH, determining binding parameters like the binding constant ( K a) and the number of binding sites ( n ). Through fluorescence binding studies, we unravelled a strong interaction between Rifaximin and RfaH. Dose-dependent quenching of intrinsic fluorescence of RfaH indicated complex formation with Rifaximin ( Fig. 2 A ). Fitting the quenching data to the modified Stern-Volmer equation yielded K a of 7.38 x 10 6 M − 1 and n of 1 ( Fig. 2 B ). This suggests a strong association of Rifaximin with a single binding site on RfaH. This robust interplay, falling within the typical range for protein-ligand complexes, implicates a potential role of Rifaximin in RfaH inhibition and related pathways, paving the way for further investigation into its antibacterial mechanisms and the development of novel therapeutic strategies. Determination of MIC and MBC for Rifaximin against KP To quantitatively assess the antibacterial efficacy of Rifaximin, we determined the MIC and MBC values against the K. pneumoniae (ATCC 700603) strain. The INT-colorimetric assay revealed a MIC of 100 µM, highlighting the potential of Rifaximin against KP. While MIC tells us the lowest concentration just prevents bacterial growth, the MBC tells us whether the agent kills the bacteria. This distinction is crucial because even if an inhibitor stops bacteria from growing, they might still be alive and potentially cause harm later. Time-kill curve experiments established an MBC of 200 µM, signifying almost complete eradication at this concentration. Notably, even at the MIC of 100 µM, Rifaximin achieved a remarkable 4-log10 reduction in bacterial population compared to the initial inoculum of 1.5 X 10 5 CFU/mL ( Fig. 3 ). These findings suggest that Rifaximin is a promising antibacterial agent against KP, warranting further investigation of its therapeutic potential, particularly in the face of multidrug-resistant strains [ 42 ]. Capsule production estimation Capsules play a significant role in bacterial virulence by enhancing drug resistance and hindering the immune system's ability to recognize surface antigens [ 43 ]. RfaH homologs are crucial regulators in diverse bacterial species (from E. coli to B. amyloliquefaciens ) controlling the expression of operons responsible for producing capsules, LPS core, antibiotics, toxins, and pili [ 44 ]. Disrupting RfaH in K. pneumoniae reduces capsule production, mirroring effects seen in E. coli due to the high similarity of their capsule biosynthesis clusters [ 45 , 46 ]. A recent study has demonstrated Eco RfaH's ability to suppress Rho-dependent termination within capsule operons, further underscoring the importance of RfaH in capsule regulation [ 47 ]. A study has investigated if Eco RfaH regions essential for E. coli gene activation are also crucial in K. pneumoniae , employing a lux reporter assay. They used the RfaH gene to delete the K. pneumoniae TOP52 strain, rendering it deficient in endogenous RfaH activity. The lux reporter utilized the Photorhabdus luminescens lux operon placed downstream of an ops element. This element is known to bind both Eco and Kpn RfaH. Notably, a similar reporter was previously used to identify key functional residues in Eco RfaH. The study findings revealed that both Eco RfaH and Kpn RfaH significantly increased lux expression (p < 0.0001 compared to vector control), mirroring their effects on LPS and capsule biosynthesis operon activation. This suggests a high degree of functional conservation in the Eco RfaH regions critical for gene activation across these bacterial species [ 15 ]. In another study mutation in the RfaH gene resulted in a severe attenuation (over 10,000-fold decrease) of the mutant strain's growth within the lungs compared to the wild-type strain. This growth defect was significantly restored by complementation with the wild-type RfaH gene, highlighting the critical role of RfaH for bacterial fitness in the lung environment. Furthermore, the RfaH mutant exhibited a smaller colony size compared to the wild type, indicative of a potential impairment in capsule biosynthesis. India ink staining revealed a substantial capsule surrounding the wild-type bacteria, while the RfaH mutant lacked a visible capsule [ 45 ]. In both E. coli and KP, the protein RfaH plays a critical role in maintaining cell envelope integrity. Deletion of RfaH in E. coli results in dramatic sensitivity to the detergent sodium dodecyl sulfate (SDS), mirroring the effect of a polar mutation within its target operon, which was responsible for lipopolysaccharide (LPS) biosynthesis [ 48 ]. This sensitivity can be alleviated by mutations in rho, a termination factor [ 49 ]. KP exhibits a similar reliance on RfaH, with its deletion leading to reduced capsule production [ 45 ]. This parallels observations in E. coli , suggesting a conserved role for RfaH in capsule biosynthesis due to the close resemblance of the corresponding gene clusters in both species [ 46 ]. Mutations within genes encoding rfaH, in K. pneumoniae , have also been linked to the development of phage resistance in this bacterium. RfaH plays an essential role in regulating and synthesizing both CPS and LPS, which are important components of the bacterial cell wall. Studies have consistently shown that mutations affecting genes involved in CPS and/or LPS synthesis contribute to phage resistance. This resistance mechanism arises from the loss of specific receptors on the bacterial surface due to the altered cell wall composition, hindering phage attachment and subsequent infection [ 50 , 51 ]. These findings highlight the crucial contribution of RfaH to cell envelope stability and its intricate interplay with other regulatory factors like Rho in both E. coli and KP. In K. pneumoniae ATCC 700603, treatment with 100 µM Rifaximin led to a dramatic reduction in capsule production, exceeding 50% compared to untreated controls ( Fig. 4 A ). To further affirm the effect of Rifaximin on KP outer capsule, 6 hours cultivated K. pnuemoniae cells were negatively stained and visualized [ 2 ]. As observed, Rifaximin treated and untreated K. pnuemoniae cells showed variation in bacterial outer capsule thickness. The untreated cells showed comparatively thicker outer capsule when compared to sub-MIC Rifaximin treated cells that showed smaller cells with relatively thin outer capsule ( Fig. 4 B ) . This observation suggests a potential role for Rifaximin in inhibiting RfaH, a critical protein in the KP capsule biosynthesis pathway. The capsule, a polysaccharide-based extracellular structure, serves as a vital shield against diverse environmental assaults, including phagocytosis by the immune system, phage infection, and desiccation [ 52 ]. Consequently, a substantial decrease in capsule content, as induced by Rifaximin, is likely to heighten the susceptibility of K. pneumoniae cells to these external stresses, potentially culminating in cell death. Further investigation is warranted to elucidate the precise mechanism by which Rifaximin interacts with RfaH and to assess the functional impact of this capsule reduction on KP virulence and its overall adaptability within the host and environment [ 53 , 54 ]. Molecular docking The molecular docking between RfaH and Rifaximin revealed a binding affinity of -9.3 kcal/mol. An in-depth analysis of the interaction between Rifaximin and the RfaH binding pocket was conducted for all 9 docked conformations of the ligand. While Rifaximin displayed interactions with RfaH at multiple sites, its favored docking position exhibited the highest binding strength when compared to alternative positions. Literature studies have demonstrated that disrupting specific contact points in Eco RfaH, namely with the β’CH (Tyr54), ops DNA (Arg73), βGL (Thr66), and S10 (Ile146), impairs RfaH-dependent gene activation in E. coli and the functional importance of these contact points extends to K. pneumoniae also. As anticipated, disrupting interactions with β'CH, S10, and ops DNA, abolishes the activity of operons regulated under RfaH. This suggests that some RfaH interaction points are universally critical for both E. coli and K. pneumoniae bacterial species [ 55 , 56 ]. Our docking results indicated that the rifaximin forms bonds with the Tyr54, Phe78, Arg80, and Asp147 residues of the RfaH protein. As Tyr54 which is a critical residue of β′ clamp helices (CH) domain and thus for RfaH activity, hence disruption of this specific contact point by rifaximin will lead to RfaH inhibition. The binding configuration of Rifaximin with RfaH is illustrated in Fig. 5 . Rifaximin binds within the binding pocket cavity of RfaH, interacting with residues situated in the β′ clamp helices (CH) domain, as depicted in Figs. 5 A and B . Figure 5 C indicates a deep penetration of Rifaximin to the pocket of RfaH. MD simulations The MD simulation is employed to gain insights into the atomic-level dynamics of protein-ligand complexes [ 57 ]. Moreover, it aids in evaluating the flexibility of the docked complex in comparison to the native protein state [ 58 ]. In this investigation, MD simulation was applied to appraise the stability of protein-ligand docked complexes, specifically those involving RfaH and Rifaximin within a water model. Employing the CHARMM36 force field, we conducted a 100-nanosecond simulation for both the RfaH and RfaH-Rifaximin complex. Detailed information on MD simulation outcomes is provided in Table 1 . Table 1 Various dynamic and structural parameters were examined and analyzed for a period of 100 ns for the RfaH-Rifaximin complex. System RMSD (nm) RMSF (nm) R g (nm) SASA (nm 2 ) #H-bonds RfaH 0.39 0.17 1.81 114.26 96 RfaH-Rifaximin 0.46 0.22 1.77 114.14 96 The protein undergoes structural dynamics upon binding to a ligand molecule. The calculation of root mean square deviation (RMSD) is a fundamental method employed to quantify the structural alterations in a protein following ligand binding. RMSD values for both the native state and the protein-ligand complex are charted across a 100 ns timeframe. The RfaH native structure exhibits slight fluctuations during this period. Notably, the time evolution of RMSD for the RfaH-Rifaximin complex displays some fluctuations after 60 ns, indicating that the system undergoes complexity and experiences minor instability during the simulation period ( Fig. 6 A ). During MD simulation, the residual flexibility of a protein over duration is assessed through a root mean square fluctuation (RMSF) plot. We have generated RMSF plots for both the RfaH native state and the RfaH-Rifaximin complex (Table 1 ). Throughout the simulation, the RfaH-Rifaximin complex exhibits slightly elevated RMSF values. Despite this, both systems demonstrate nearly synchronized RMSF distributions ( Fig. 6 B ) . This implies that the protein-ligand complex maintains stability post-binding. The RMSF analysis reveals minimal differences in RMSF between the two systems, signifying a stable complex. In MD simulation, the evaluation of the structural folding and conformational dynamics of a protein involves estimating the radius of gyration ( R g). The R g is computed by determining the average distance of each atom from the center of mass of the protein molecule, employing the square of each atom's distance. Throughout the simulation, variations in the size and shape of the protein molecule are observed through the R g, providing insights into the stability of the protein. Throughout a 100 ns duration, we computed the R g values for both the RfaH native structure and the RfaH-Rifaximin complex ( Fig. 7 A ) . There is not any significant difference in the R g values between both systems. By the 100 ns mark, the R g values for both the free protein and the complex converge. This convergence suggests that RfaH, upon binding to Rifaximin, exhibits stable conformational dynamics and folding. The solvent-accessible surface area (SASA) indicates the portion of the protein's surface that is accessible to solvent molecules. Assessing the solvent-accessible surface area is a pivotal technique for evaluating interactions between a protein and a ligand. This measure is valuable in evaluating the stability of the protein-ligand complex and identifying potential binding sites. The analysis of SASA is widely used to understand the stability and folding properties of proteins and protein-ligand complexes. We have graphed the SASA values for both the RfaH native structure and the RfaH-Rifaximin complex throughout the entire simulation duration ( Fig. 7 B ). The mean SASA value for both systems remains constant, exhibiting no significant alteration. Throughout the simulation period, no notable distinctions are observed, and the values converge by 100 ns. This convergence implies a stable structural folding and dynamics of the protein upon binding to the ligand. Dynamics of hydrogen bonds The stability of the protein-ligand complex relies on the establishment of hydrogen bonds [ 59 ]. Intra-molecular hydrogen bonds were calculated for both the RfaH native structure and RfaH after binding with Rifaximin, and the results were plotted over 100 ns duration ( Fig. 8 A ) . The plot generated indicates that there were no significant alterations observed in the hydrogen bonding interactions within the RfaH protein upon the formation of a complex with Rifaximin. The plot displays a constant number of hydrogen bonds for both systems. Intermolecular hydrogen bonds were also estimated to infer the stability of interactions of RfaH with Rifaximin. These interactions revealed the formation of up to six hydrogen bonds, with four consistently present bonds throughout the trajectory ( Fig. 8 B ). Evaluation of secondary structures Examining the dynamics of the secondary structure content of protein is a means to understand its conformational behavior and folding mechanism [ 60 ]. We calculated the alterations in the secondary structure for RfaH when bound to Rifaximin. The structural elements in the unbound RfaH exhibit nearly constant and equilibrated characteristics throughout the 100 ns simulation period ( Fig. 9 A ). However, a small decrease in the β-sheets and a slight increase in α-helix and content of RfaH can be seen upon compound binding ( Fig. 9 B ) . The average number of residues engaged in secondary structure formation differs in the case of the RfaH-Rifaximin complex compared to free RfaH (Table 2 ). Despite this, there is no major change observed in the secondary structure of RfaH upon the binding of Rifaximin, indicating strong stability of the complex. Table 2 Evaluation of Secondary Structures during MD simulation of RfaH native and RfaH-Rifaximin complex RfaH RfaH-Rifaximin Coil 36 38 β-sheet 37 34 β-bridge 1 2 Bend 15 15 Turn 17 16 α-helix 53 56 3 10 -helix 2 0 PCA and FEL analysis The PCA is a crucial technique for assessing the collective motion of atoms in a protein-ligand complex [ 61 ]. PCA is employed to analyze the conformational changes in both the native RfaH and the RfaH-Rifaximin complex, utilizing projections of Cα atoms to estimate the conformational dynamics of these systems. The plots illustrating these analyses are presented in Fig. 10 A. The subspaces occupied by free RfaH closely align with those of the protein-ligand complexes. The native RfaH occupies one subspace, while the complex occupies two subspaces, signifying a reduction in stability within the complex. The protein-ligand systems show some variability concerning the free state of the protein. Assessing the protein folding mechanism involves the utilization of Free Energy Landscape (FEL) analysis. This analysis is employed to determine global and local minima points within the energy landscape of a protein. The FEL plots for both the unbound RfaH and the RfaH-Rifaximin complex are depicted in Figs. 10 B and C . Regions in deep blue color within the plots represent low-energy states, closely associated with the native states. The free state of the protein showed one large basin. In the case of RfaH-Rifaximin three distinct basins are formed. It suggests that the global minimum of free RfaH is slightly disturbed by the binding of Rifaximin. In summary, the FEL analysis suggests that the interaction of Rifaximin with RfaH does not induce protein unfolding throughout the 100 ns timeframe. Discussion The alarming rise of antimicrobial resistance (AMR) in KP necessitates a multi-pronged approach, including judicious antibiotic use, enhanced infection control, and continual exploration of novel therapeutic strategies. Repurposing existing drugs, for example, the rapid FDA approval of Remdesivir for COVID-19 [ 41 ], presents a promising avenue for combating KP infections. Rifaximin shows broad-spectrum bactericidal activity against various enteric pathogens. Encompassing both gram-positive and negative, aerobic and anaerobic bacteria, this efficacy extends to a diverse range of enterotoxic and pathogenic species. A comprehensive microbiological survey conducted revealed a minimum inhibitory concentration (MIC90) ranging from 4 to 64 µg/ml for enteric pathogens like Escherichia coli, Salmonella, Shigella, Campylobacter, Plesiomonas, and Aeromonas , isolated across three continents [ 62 ]. These findings align with observations from other studies, solidifying the consistent susceptibility patterns of these bacteria towards Rifaximin [ 63 ]. Notably, Rifaximin readily achieves concentrations exceeding 8,000 µg/g within human feces, effectively surpassing the MIC90 values and ensuring potent action against these enteric pathogens within the gastrointestinal tract [ 64 ]. While Rifaximin boasts high gut concentrations and a broad spectrum of antibacterial activity, it exhibits a surprising lack of significant disruption to the intestinal microbiota. A two-week course of Rifaximin in human subjects resulted in just a 1 log reduction in intestinal coliforms per gram of stool, highlighting its minimal impact on the overall gut microbial population [ 65 ]. Due to its minimal systemic absorption, Rifaximin also has a favorable safety profile with a low occurrence of adverse events. In clinical trials involving over 1000 participants, those receiving Rifaximin reported side effects at frequencies comparable to or even lower than those observed in groups given placebo, ciprofloxacin, or TMP-SMX, further solidifying its reputation as a well-tolerated medication [ 66 ]. These characteristics make Rifaximin a valuable therapeutic option offering a distinct advantage over traditional antibiotics, which often cause substantial collateral damage to the beneficial gut microbiome and mitigating potential side effects. Rifamixin is a structural analog of Rifampicin [ 67 ]. We have done in-silico studies showing both Rifampicin and Rifamixin share the same binding pocket on DNA-dependent RNA polymerase (Supplementary Figure S1 ). A recent study has suggested that Rifampicin binds to the beta subunit ( rpoB ) of DNA-dependent RNA polymerase in hypermucoviscous strain of Klebseilla pneumoniae that reduces its mucoviscosity. However, there is a persistent mystery regarding its mechanism of action as the authors do not believe that sterically blocked Rifampicin-bound RNA polymerase plays an active role in the anti-mucoviscouscos mechanism i.e. reducing capsule production [ 68 ]. Mutations in the rpoB of RNA polymerase, leads to alteration in the phenotypic traits of bacterial cells by affecting transcription elongation and Rho-dependent and Rho-independent termination [ 69 ]. In E.coli rho mutants showed changes in the expression of rfaH genes forming altered outer membrane or capsule production [ 70 ]. The overall decrease in transcriptional activity leads to change in the gene expression pattern by inducing local and global cellular responses. The sub-MIC levels of antibiotics irrespective of their mode of action show transcriptional modulatory effects [ 71 , 72 ]. In the case of Salmonella typhimurium , many antimicrobials modulate the transcription of subset genes. These antimicrobials are of different structures and have different modes of action [ 73 ]. A detailed study involving Rifampicin and Salmonella Typhimurium transcriptional modulations has been done. The study reveals that many subsets of genes are being down-regulated as well as up-regulated as an effect of sub-MIC levels of Rifampicin. In spite of an in-depth analysis of the authors regarding the divergently affected promoter the precise mechanism remains unclear for their observations [ 74 ]. Based on these studies, we can speculate that as seen in the Salmonella Typhimurium and its global transcriptional changes may happen in Klebseilla pnuemoniae when subjected to sub-MIC levels of Rifamixin leading to changes in the gene expression of RfaH and hence capsule production. Our study provides a foundation for the potential repurposing of rifaximin in the fight against KP infections. While its limited absorption within the gut presents a potential hurdle in future in-vivo studies, our in-vitro findings demonstrate its antibacterial activity against KP through its interaction with the RfaH protein. This crucial discovery opens avenues for developing novel RfaH inhibitors with improved gut absorption properties based on the structural framework of Rifaximin. By synergistically integrating insights from disease pathophysiology, drug pharmacology, and computational analysis, we can prioritize promising drug candidates for KP infections. This interdisciplinary approach holds immense potential for accelerating the development of effective KP therapies, ultimately improving patient outcomes and mitigating the healthcare burden. Conclusions In light of the rising threat posed by multidrug-resistant KP, repurposing existing FDA-approved drugs emerges as a promising strategy. This study explores the potential of rifaximin, a non-absorbable oral antibiotic currently used for irritable bowel syndrome (IBS) [ 75 ], as a novel therapeutic against KP infections. Our exploration of the drug repurposing potential of rifaximin as a targeted therapeutic against KP, leveraging the inhibition of the transcription anti-termination protein RfaH, reveals a promising avenue for novel antimicrobial development. The remarkable antimicrobial activity demonstrated by Rifaximin, coupled with its significant reduction in capsule production in KP, suggests its efficacy in obstructing the crucial activities of RfaH. A strong binding affinity of Rifaximin to RfaH evidenced by fluorescence studies, and the stable MD simulation data further elucidate the intricate and sustained interaction between rifaximin and RfaH. These findings collectively emphasize the multifaceted potential of rifaximin as a repurposed antibiotic that can disrupt key virulence mechanisms in KP. However, moving forward, a rigorous exploration and validation of rifaximin's potential in clinical settings are imperative to implicate its full therapeutic capabilities and revolutionize novel approaches to combat bacterial infections. Abbreviations KP: Klebsiella pneumoniae, MDR-KP: Multidrug-resistant Klebsiella pneumoniae , R g : Radius of gyration, MIC: Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration, MD: Molecular Dynamics, RMSD: Root Mean Square Deviation, RMSF: Root Mean Square Fluctuation, SASA: Solvent Accessible Surface Area Declarations Disclosure statement No potential conflict of interest was reported by the author(s). Data Availability Statement: The information supporting this study is available in this article. Funding: This work is funded by the Indian Council of Medical Research (Grant No. ECD/adoc/2/2021-22). Acknowledgements: AA thanks to Indian Council of Medical Research-Department of Health Research (File No. R.12014/06/2022-HR) for financial support. MIH thanks the Indian Council of Medical Research for financial support (Grant No. ECD/adoc/2/2021-22). Credit author statement: Anam Ashraf : Conceptualization, Investigation, Methodology, Writing- Original draft preparation, Arunabh Chaudhary : Data curation, Graphics, Data validation; Mohammad Ali Khan: Methodology, Data validation, Writing-review, and editing; Saba Noor: Graphics, Data validation, Writing-review, and editing; Asimul Islam: Data curation, Graphics, Data validation; Md. Imtaiyaz Hassan: Conceptualization, Investigation, Supervision, Review and editing, and project administration. References Karampatakis T, Tsergouli K, Behzadi P (2023) Carbapenem-resistant Klebsiella pneumoniae: Virulence factors, molecular epidemiology and latest updates in treatment options. Antibiotics 12:234 Russo A, Fusco P, Morrone HL, Trecarichi EM, Torti C (2023) New advances in management and treatment of multidrug-resistant Klebsiella pneumoniae. 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Res Microbiol 164:416–424 Nanda S (2011) Rifaximin provides effective and sustained relief of IBS symptoms. Nat Reviews Gastroenterol Hepatol 8:121–121 Additional Declarations No competing interests reported. Supplementary Files FigureS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4724428","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326425008,"identity":"979afdd5-9268-4a0f-ab7e-07880da87bd9","order_by":0,"name":"Anam Ashraf","email":"","orcid":"","institution":"Jamia Millia Islamia","correspondingAuthor":false,"prefix":"","firstName":"Anam","middleName":"","lastName":"Ashraf","suffix":""},{"id":326425009,"identity":"af0b3bc7-b6fa-47f7-9f54-9e800c6287ef","order_by":1,"name":"Arunabh Choudhary","email":"","orcid":"","institution":"Jamia Millia Islamia","correspondingAuthor":false,"prefix":"","firstName":"Arunabh","middleName":"","lastName":"Choudhary","suffix":""},{"id":326425010,"identity":"ae1287ef-0415-4a9b-8fad-c2f9aad99da9","order_by":2,"name":"Mohammad Ali Khan","email":"","orcid":"","institution":"Jamia Hamdard","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Khan","suffix":""},{"id":326425011,"identity":"c2833209-cdbe-418b-a148-3300c38b227e","order_by":3,"name":"Saba Noor","email":"","orcid":"","institution":"Jamia Millia Islamia","correspondingAuthor":false,"prefix":"","firstName":"Saba","middleName":"","lastName":"Noor","suffix":""},{"id":326425012,"identity":"83101824-4d56-4e67-81d0-cf4fe7478dab","order_by":4,"name":"Asimul Islam","email":"","orcid":"","institution":"Jamia Millia Islamia","correspondingAuthor":false,"prefix":"","firstName":"Asimul","middleName":"","lastName":"Islam","suffix":""},{"id":326425013,"identity":"ab45c69a-7917-40f8-88d8-63948e60f454","order_by":5,"name":"Md. Imtaiyaz Hassan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYDACZh4Izc9wAAiBgI1oLZINRGthgGoxOECsu3TbeQ9+/PHHLs/44OnEAww1dgx80g34tZgd5kuW5m1LLjY7cHbDAYZjyQxsMgTsMzvMYyDN2MCcuA2shQ2IJBIIajH++eNPfeLmBpCWf8RpMZPgYTucuIEBqIWxjUgt1rxtxxNngByW2JfMQ1jL+TPGN3/8qU7sn3F284cP3+zk5GcQ0IIAEgcYGBLg0UQU4G8gQfEoGAWjYBSMKAAAX/lGe1i8t9IAAAAASUVORK5CYII=","orcid":"","institution":"Jamia Millia Islamia","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Imtaiyaz","lastName":"Hassan","suffix":""}],"badges":[],"createdAt":"2024-07-11 13:23:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4724428/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4724428/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61953625,"identity":"662c778e-b76b-4c3f-b683-a2471ab5b13c","added_by":"auto","created_at":"2024-08-07 13:08:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1527925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCloning, expression and purification of RfaH A) \u003c/strong\u003eRestriction digestion of the positive colonies showing positive fallout \u003cstrong\u003eB)\u003c/strong\u003e Sequencing data of the positive clone \u003cstrong\u003eC)\u003c/strong\u003e SDS-PAGE profile showing a single band of RfaH.\u003c/p\u003e","description":"","filename":"floatimage131.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/896ac62427ced834e36e2cd7.png"},{"id":61953617,"identity":"c8ea7c3f-63cf-4a62-bd14-95f483796a6f","added_by":"auto","created_at":"2024-08-07 13:08:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":613567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence binding studies of Rifaximin with RfaH. (A)\u003c/strong\u003e Fluorescence emission spectra of RfaH with increasing concentrations of Rifaximin. RfaH was excited at 280 nm, and the emission spectra were recorded in the range of 300-400 nm. \u003cstrong\u003e(B)\u003c/strong\u003e A modified Stern-Volmer plot was used to analyze the quenching data of Rifaximin (0-10.489 μM) quantitatively and to determine the binding constant (\u003cem\u003eKa\u003c/em\u003e) and the number of binding sites (\u003cem\u003en\u003c/em\u003e) for Rifaximin.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/e166a94b3a917db2868c7e4a.png"},{"id":61953622,"identity":"891d36cb-cc99-4898-9321-bf7e3d46a3a0","added_by":"auto","created_at":"2024-08-07 13:08:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime kill assay for Rifaximin against KP – \u003c/strong\u003eThe bactericidal kinetics of Rifaximin against actively growing \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603) using a range of concentrations (0.5X MIC to 5X MIC) were conducted. Consistent results were obtained across three replicates, with the presented data representing a typical outcome.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/e1db42c742bd0620f91af0d7.png"},{"id":61953626,"identity":"9f84ab6c-25b4-4e0a-8683-49ac76595cc8","added_by":"auto","created_at":"2024-08-07 13:08:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCapsule production in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eK. pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with and without Rifaximin treatment. A)\u003c/strong\u003e The capsule production was estimated. The experiment is conducted thrice with consistent results. The presented data represents a typical outcome. B) Microscopic evaluation of the capsule visualized using Nigrosin dye staining. Representative images for the \u003cem\u003eK. pneumoniae\u003c/em\u003e cells untreated and C) Rifamixin treated. Bar represents 10mm scale.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/92cdbc880f7ad0e45cfc27e2.png"},{"id":61953628,"identity":"d5a1c7aa-f84a-4719-a72b-d815c9337fd8","added_by":"auto","created_at":"2024-08-07 13:08:49","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":413785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteractions of Rifaximin with RfaH\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e 2-D Interactions of Rifaximin with binding pockets of RfaH \u003cstrong\u003e(B)\u003c/strong\u003e 3-D Interactions of Rifaximin with binding pockets of RfaH \u003cstrong\u003e(C)\u003c/strong\u003e Surface representation of substrate-binding pocket of RfaH in complex with Rifaximin.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/3dd9154b51b0431324fdffae.jpeg"},{"id":61953621,"identity":"bade2acd-1c4f-4bd2-925f-0de4db680d42","added_by":"auto","created_at":"2024-08-07 13:08:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":296452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRMSD and RMSF of the native RfaH protein and RfaH-Rifaximin complex\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e RMSD of the native form of RfaH is shown in black and RfaH with Rifaximin is shown in red. \u003cstrong\u003e(B)\u003c/strong\u003e RMSF of the native form of RfaH is shown in black and RfaH with Rifaximin is shown in red. Lower panel shows showing principal density function (PDF) of RMSD and RMSF, respectively.\u003c/p\u003e","description":"","filename":"floatimage65.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/b9ffef73491ad7c2a635f3a7.png"},{"id":61954260,"identity":"e5623776-4782-4907-bb8d-5b864797eee4","added_by":"auto","created_at":"2024-08-07 13:16:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":357199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural Compactness of RfaH. (A)\u003c/strong\u003e \u003cem\u003eR\u003c/em\u003eg plot of RfaH and RfaH-Rifaximin complex with native represented in black color whereas the complex is represented in red color. (\u003cstrong\u003eB)\u003c/strong\u003e SASA plot Rg plot of RfaH and RfaH-Rifaximin complex. The lower panel shows the principal density function (PDF) of \u003cem\u003eR\u003c/em\u003eg and SASA, respectively.\u003c/p\u003e","description":"","filename":"floatimage71.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/8571da62f5c2c360ca930a57.png"},{"id":61953631,"identity":"f3829423-10f7-40d2-80be-10d7ace909bd","added_by":"auto","created_at":"2024-08-07 13:08:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":303504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH-bond interaction counts during MD simulation. A) \u003c/strong\u003eH-bond interaction counts of RfaH interactions with Rifaximin. \u003cstrong\u003eB)\u003c/strong\u003e Intermolecular hydrogen interactions count for the RfaH-Rifaximin complex. The lower panel shows the principal density function (PDF) of H-bond interactions.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/f0e8189009b39393a1a50e5e.png"},{"id":61953618,"identity":"e3985c59-18ae-4c05-ab45-031accd36127","added_by":"auto","created_at":"2024-08-07 13:08:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":415414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of secondary structures\u003c/strong\u003e of \u003cstrong\u003e(A)\u003c/strong\u003e RfaH and \u003cstrong\u003e(B) \u003c/strong\u003eRfaH-Rifaximin complex during 100 ns MD simulation.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/57432e7b2237ab3d329d764e.png"},{"id":61953627,"identity":"fc5cade0-3adb-400c-abbf-be5e737f3a72","added_by":"auto","created_at":"2024-08-07 13:08:48","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":738886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePCA analysis of native RfaH and RfaH-Rifaximin complex. FEL analysis of \u003cstrong\u003e(B)\u003c/strong\u003eNative RfaH and \u003cstrong\u003e(C)\u003c/strong\u003e RfaH-Rifaximin complex.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/4e858b742bd3ec3df2ee13e1.jpeg"},{"id":62310587,"identity":"aed3086e-57d8-4069-8e40-f7c1623baedd","added_by":"auto","created_at":"2024-08-12 20:11:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5833233,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/2dc0a38c-857a-4ac8-bdec-f0c2b95cdaa5.pdf"},{"id":61953623,"identity":"6b81b582-47e7-4c36-852f-c7c344c945a4","added_by":"auto","created_at":"2024-08-07 13:08:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1198515,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4724428/v1/f90b0a3c66f8f26936a3ac4a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Repurposing Rifaximin against Klebsiella pneumoniae via Targeting of Transcription Anti-termination Protein RfaH for Novel Antimicrobial Development","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (KP), a ubiquitous Gram-negative bacterium, is a significant cause of community and healthcare-associated infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Multidrug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (MDR-KP) has emerged as a formidable threat to global health, posing a significant challenge in the field of infectious diseases. This bacterium is notorious for its ability to evade the effects of a wide range of antibiotics, rendering treatment options scarce and compromising patient outcomes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The rising prevalence of antimicrobial resistance (AMR) in KP poses a substantial problem to healthcare systems worldwide due to its alarming resistance to multiple classes of antibiotics, including aminoglycosides, fluoroquinolones, cephalosporins, and even carbapenems, considered the last-line of defense against bacterial infections [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The alarming rise of multidrug-resistant (MDR) and hypervirulent (hv) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (MDR-hvKp) convergent clones necessitates active surveillance networks and robust monitoring systems to track and manage the spread of these highly successful hybrid pathogens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrug repurposing is a powerful and rapidly evolving strategy that offers a promising avenue for accelerating drug discovery and development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It aims to address the lengthy and resource-intensive nature of conventional drug development. By focusing on rationally understanding and redefining the therapeutic potential of existing drugs, we can leverage the established safety profiles and bypass the extensive clinical trial phases associated with new drug development. This approach resonates with the growing trend in drug discovery, where computational methods are increasingly employed to rapidly identify promising targets for either the repurposing of existing drugs or the rational design of novel chemical entities, accelerating the fight against emerging infectious diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Its numerous advantages, including reduced cost, faster development times, and established safety profiles can be particularly beneficial in addressing multidrug-resistant infections or targeting complex diseases characterized by multiple underlying mechanisms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Rifaximin is an orally administered antibiotic with minimal systemic absorption. It possesses broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Rifaximin, a non-aminoglycoside antibiotic is a semisynthetic derivative of the natural antibiotic Rifamycin. It is a structural analog of rifampin, another antibiotic derived from Rifamycin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The irreversible nature of Rifaximin binding to RNA polymerase ensures sustained inhibition of bacterial RNA synthesis, providing a prolonged therapeutic effect. This characteristic contributes to the effectiveness of rifaximin in treating various bacterial infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRfaH, essential for virulence in pathogens like \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, activates virulence genes through intricate interactions with RNA polymerase and ribosomes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This dual action promotes transcription and translation of key operons involved in capsule, cell wall, and pilus biosynthesis, ultimately enhancing bacterial virulence [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the past decades, researchers have uncovered a group of operons whose full function relies on the protein RfaH in various bacterial strains. These operons are responsible for producing vital components of bacteria, including the protective LPS core [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the O-antigen on the surface [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the α-hemolysin toxin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the F-factor for genetic exchange [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the hemin receptor for iron uptake [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and even the group I, II, and III capsules that shield the bacteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. When RfaH is absent, the expression of these operons diminishes, leading to a significant decrease in the levels of these vital components, ultimately affecting bacterial viability, virulence, and interaction with the host immune system [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. RfaH acts as an anti-terminator, counteracting the effect of terminators and allowing for the complete transcription of operons. Understanding RfaH's role in suppressing operon polarity provides valuable insights into gene regulation in bacteria [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This, in turn, targeting RfaH can have a significant impact on bacterial health and virulence, affecting their ability to withstand environmental stress, evade the immune system, and cause disease [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the critical role of RfaH in KP survival and virulence, targeting this protein with repurposed drugs presents an attractive therapeutic strategy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Rifaximin, a known antibacterial agent, emerged as a promising candidate in this study. Utilizing in vitro assays and spectroscopic techniques, we established the inhibitory effect of Rifaximin on KP, likely through its interaction with RfaH. Furthermore, molecular dynamics (MD) simulations corroborated our experimental findings, lending additional support to the potential of rifaximin as a repurposed therapeutic option for KP infections. These results warrant further investigation of the efficacy of rifaximin and the mechanism of action against KP, potentially leading to the development of a novel therapeutic approach for combating this critical pathogen.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCloning, Expression and Purification of RfaH\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eRfaH\u003c/em\u003e gene of \u003cem\u003eKlebseilla pneumoniae\u003c/em\u003e was cloned commercially using gene-specific primers with 6X His tag (GenScript Biotech, NJ, USA). The protein was expressed in \u003cem\u003eE. coli\u003c/em\u003e strain BL21 expression cells and purified using the Ni-NTA column. Briefly, the construct of RfaH was PCR-amplified and cloned into the pET28b(+) vector. Restriction digestion and DNA sequencing verified the resulting clone. For maximum expression, RfaH was induced with 0.5 mM IPTG and incubated for 16 hours at 16\u0026deg;C post-induction. The cells were then lysed using a sonicator in lysis buffer (Tris 20mM NaCl 200mM PMSF 7.5mM and DTT 0.1mM). The resulting lysate was loaded on the Ni-NTA column and purified using an imidazole concentration gradient. The purified RfaH was eluted and analyzed on SDS-PAGE and confirmed by mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence measurements\u003c/h2\u003e \u003cp\u003eA fluorescence quenching experiment was performed on a Jasco spectrofluorometer (FP 8200, Japan) to assess the interaction of rifaximin with RfaH. Rifaximin was first dissolved in DMSO to generate a 50 mM stock solution, which was subsequently diluted to a working concentration of 0.5 mM using a Tris-NaCl buffer. With a fixed concentration of RfaH at 8 \u0026micro;M, rifaximin was titrated in a 1:9 ratio until saturation. Emission spectra were recorded within the 300\u0026ndash;400 nm range upon excitation of tryptophan at 280 nm. Slit widths and sensitivity settings were standardized for all measurements. The obtained data was analyzed using the modified Stern-Volmer equation to determine the binding affinity (\u003cem\u003eK\u003c/em\u003ea) and number of binding sites (\u003cem\u003en\u003c/em\u003e) for rifaximin on RfaH. Triplicate measurements were conducted for each experiment, with appropriate corrections applied for buffer blanks and inner filter effects. This comprehensive approach ensured an accurate and reliable evaluation of the interaction between rifaximin and RfaH using our well-established protocol [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration measurements\u003c/h2\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) of rifaximin against \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 7006043) using the INT (p-iodonitrotetrazolium chloride) colorimetric assay was performed. Rifaximin was dissolved in DMSO, ensuring its concentration remained below 2.5% to avoid affecting bacterial growth. The test solution was serially diluted in broth media in a 96-well plate, followed by the addition of 100 \u0026micro;L inoculum containing 1.5 x 10\u003csup\u003e5\u003c/sup\u003e CFU/mL of bacteria per well. The plate was sealed, shaken, and incubated at 37\u0026deg;C for 18 hours. Negative controls included KP cultures without any inhibitor and media alone. A positive control involved KP cultured with ciprofloxacin, a known antibiotic. The assay was repeated three times for accuracy. After incubation, 0.2 mg/mL INT was added to each well, and the plate was further incubated for 30 minutes. Viable bacteria reduced the yellow dye to pink, indicating their presence. The MIC was defined as the lowest concentration of rifaximin that prevented color change and completely inhibited bacterial growth [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of minimum bactericidal concentration\u003c/h2\u003e \u003cp\u003eTime-killing curves were employed to explore the bactericidal effects of Rifaximin against \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700634). Freshly prepared bacteria in their early logarithmic growth phase were exposed to Rifaximin at various concentrations, ranging from 0.5 to 5 times the MIC. This range aimed to capture both potential killing and stalling effects, as the MIC might only halt growth without reducing the initial bacterial count. Controls included bacteria without inhibitors (growth control) and media alone (sterility control). At specific time points (3, 6, 9, 12, and 24 hours), samples were taken, diluted, plated, and incubated for 24 hours to count the remaining colony-forming units (CFUs). The CFU data was then plotted on a logarithmic scale (y-axis) against time (x-axis) to visualize the killing activity of Rifaximin over time. Each experiment was repeated three times for statistical accuracy. This setup allowed researchers to determine the time-dependent killing effectiveness of Rifaximin against KP [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCapsule quantification assay and visualization\u003c/h2\u003e \u003cp\u003eTo investigate the impact of Rifaximin on KP capsular polysaccharide (CPS) production and uronic acid content we did a capsule quantification assay explained elsewhere [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Briefly, 20 ml LB broth cultures with and without 100\u0026micro;M Rifaximin were inoculated with \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603) and incubated for 16 hours. Cell density was normalized by CFU/ml determination. A modified Zwittergent extraction protocol was employed as follows [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], 500 \u0026micro;L cultures were incubated with 1% Zwittergent in 100mM citric acid at 50\u0026deg;C for 20 min and then centrifuged at 10,000g for 5 min. The supernatants were precipitated with cold ethanol by incubating it at 4\u0026deg;C for 20 min. After centrifugation at 10,000g for 5 min the pellets were resuspended with water and treated with 12.5 mM sodium tetraborate in concentrated sulfuric acid. The samples were then boiled at 95\u0026deg;C for 5 min and reacted with 0.15% 3-phenylphenol in 0.5% NaOH. Absorbance at 520nm was measured, normalized to CFU/ml, and averaged from triplicate assays, providing a quantitative measure of uronic acid content relative to bacterial density. This approach allowed a comparison of CPS production and uronic acid content in KP with and without Rifaximin treatment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe outer capsule of the cells was visualized by negative staining using log phase \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603) culture, which was mixed with 7% nigrosin dye. Rifamixin-treated and untreated cells were visualized under the microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eTo unveil the interactions between the KP RfaH protein and the antibiotic Rifaximin molecular docking was done. The 3D structure of RfaH was modeled from AlphaFold (ID: AF-W1HRW8-F1). Subsequently, the structure was refined through refinement processes utilizing Swiss PDB Viewer and MGL tools. MGL tools added hydrogen atoms and assigned Kollman charges to RfaH's polar groups, mimicking its real-life chemistry. InstaDock [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] was used to virtually dock Rifaximin onto RfaH, exploring numerous binding possibilities like an exhaustive eight-round search. Finally, Discovery Studio Visualizer [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and PyMOL [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] were used to analyze the resulting protein-ligand complexes, revealing the intricate interactions between RfaH and Rifaximin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMD simulations\u003c/h2\u003e \u003cp\u003eMD simulation stands as a crucial in-silico technique for understanding the structural dynamics and interactions between a ligand and a protein molecule [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The stability and structural dynamics of the RfaH protein bound to Rifaximin were assessed through a simulation lasting 100 ns. The simulation of both the RfaH-Rifaximin complex and the native RfaH protein was conducted using GROMACS version 5.5.1 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Two configurations were established for the protein and protein-ligand complex. In each configuration, a cubic box was generated, maintaining a 10\u0026Aring; distance from the protein and incorporating the simple point-charge (SPC16) water model. To eliminate potential steric hindrance among atoms, an energy minimization technique was applied to the solvated systems. This involved 1500 steps of the steepest descent method for energy minimization. A two-step equilibration lasting 100 ps, incorporating periodic boundary conditions, was subsequently conducted. Following this, a simulation of 100 nanoseconds was executed for each configuration, and the resultant trajectories were analyzed utilizing the 'gmx' tools [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe alarming rise of antimicrobial resistance (AMR) in KP necessitates a multi-pronged approach, including judicious antibiotic use, enhanced infection control, and continual exploration of novel therapeutic strategies [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Repurposing existing drugs, for example, the rapid FDA approval of Remdesivir for COVID-19 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], presents a promising avenue for combating KP infections. Our study lays the groundwork for repurposing Rifaximin against KP infections. While its poor gut absorption may pose challenges in in-vivo studies our in-vitro data demonstrate its antibacterial activity against KP. This paves the way for designing novel RfaH inhibitors based on rifaximin scaffold, potentially with improved gut absorption. By synergistically integrating insights from disease pathophysiology, drug pharmacology, and computational analysis, we can prioritize promising drug candidates for KP infections. This interdisciplinary approach holds immense potential for accelerating the development of effective KP therapies, ultimately improving patient outcomes and mitigating the healthcare burden.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCloning, expression and purification of RfaH\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eRfaH\u003c/em\u003e gene was cloned into pET-28a(+) vector using gene-specific primers with 6X His tag. The positive colonies were confirmed by using restriction digestion enzymes \u003cem\u003eNde\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e The plasmid was then transformed into DH5α to increase the copy number and in BL21 (DE3) cells for expression. The positive clones were screened using a specific antibiotic kanamycin, which was further confirmed by sequencing \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e His-tagged recombinant RfaH protein was purified using Ni-NTA affinity chromatography. Eluted fractions were analyzed by SDS-PAGE depicting a single purified band \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence binding studies\u003c/h2\u003e \u003cp\u003eTo investigate the antibacterial effect of Rifaximin through RfaH inhibition, we employed fluorescence binding studies. The aromatic residues, particularly tryptophan, tyrosine, and phenylalanine, have unique fluorescence properties that are influenced by factors like solvent polarity, temperature, and interactions with other molecules. When a ligand binds to a protein, it can alter the local environment around these aromatic residues, leading to changes in their fluorescence emission intensity, spectra, or lifetime. This helps to monitor the formation and dynamics of protein-ligand complexes in real-time, providing valuable insights into binding affinity, specificity, and potential mechanisms of action.\u003c/p\u003e \u003cp\u003eWe used a quenching approach to assess the interaction between Rifaximin and RfaH, determining binding parameters like the binding constant (\u003cem\u003eK\u003c/em\u003ea) and the number of binding sites (\u003cem\u003en\u003c/em\u003e). Through fluorescence binding studies, we unravelled a strong interaction between Rifaximin and RfaH. Dose-dependent quenching of intrinsic fluorescence of RfaH indicated complex formation with Rifaximin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e Fitting the quenching data to the modified Stern-Volmer equation yielded \u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e of 7.38 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003en\u003c/em\u003e of 1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e This suggests a strong association of Rifaximin with a single binding site on RfaH. This robust interplay, falling within the typical range for protein-ligand complexes, implicates a potential role of Rifaximin in RfaH inhibition and related pathways, paving the way for further investigation into its antibacterial mechanisms and the development of novel therapeutic strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of MIC and MBC for Rifaximin against KP\u003c/h2\u003e \u003cp\u003eTo quantitatively assess the antibacterial efficacy of Rifaximin, we determined the MIC and MBC values against the \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603) strain. The INT-colorimetric assay revealed a MIC of 100 \u0026micro;M, highlighting the potential of Rifaximin against KP. While MIC tells us the lowest concentration just prevents bacterial growth, the MBC tells us whether the agent kills the bacteria. This distinction is crucial because even if an inhibitor stops bacteria from growing, they might still be alive and potentially cause harm later. Time-kill curve experiments established an MBC of 200 \u0026micro;M, signifying almost complete eradication at this concentration. Notably, even at the MIC of 100 \u0026micro;M, Rifaximin achieved a remarkable 4-log10 reduction in bacterial population compared to the initial inoculum of 1.5 X 10\u003csup\u003e5\u003c/sup\u003e CFU/mL \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e These findings suggest that Rifaximin is a promising antibacterial agent against KP, warranting further investigation of its therapeutic potential, particularly in the face of multidrug-resistant strains [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCapsule production estimation\u003c/h2\u003e \u003cp\u003eCapsules play a significant role in bacterial virulence by enhancing drug resistance and hindering the immune system's ability to recognize surface antigens [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. RfaH homologs are crucial regulators in diverse bacterial species (from \u003cem\u003eE. coli\u003c/em\u003e to \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e) controlling the expression of operons responsible for producing capsules, LPS core, antibiotics, toxins, and pili [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Disrupting RfaH in \u003cem\u003eK. pneumoniae\u003c/em\u003e reduces capsule production, mirroring effects seen in \u003cem\u003eE. coli\u003c/em\u003e due to the high similarity of their capsule biosynthesis clusters [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. A recent study has demonstrated Eco RfaH's ability to suppress Rho-dependent termination within capsule operons, further underscoring the importance of RfaH in capsule regulation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA study has investigated if Eco RfaH regions essential for \u003cem\u003eE. coli\u003c/em\u003e gene activation are also crucial in \u003cem\u003eK. pneumoniae\u003c/em\u003e, employing a lux reporter assay. They used the RfaH gene to delete the \u003cem\u003eK. pneumoniae\u003c/em\u003e TOP52 strain, rendering it deficient in endogenous RfaH activity. The lux reporter utilized the Photorhabdus luminescens lux operon placed downstream of an ops element. This element is known to bind both Eco and Kpn RfaH. Notably, a similar reporter was previously used to identify key functional residues in Eco RfaH. The study findings revealed that both Eco RfaH and Kpn RfaH significantly increased lux expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 compared to vector control), mirroring their effects on LPS and capsule biosynthesis operon activation. This suggests a high degree of functional conservation in the Eco RfaH regions critical for gene activation across these bacterial species [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn another study mutation in the RfaH gene resulted in a severe attenuation (over 10,000-fold decrease) of the mutant strain's growth within the lungs compared to the wild-type strain. This growth defect was significantly restored by complementation with the wild-type RfaH gene, highlighting the critical role of RfaH for bacterial fitness in the lung environment. Furthermore, the RfaH mutant exhibited a smaller colony size compared to the wild type, indicative of a potential impairment in capsule biosynthesis. India ink staining revealed a substantial capsule surrounding the wild-type bacteria, while the RfaH mutant lacked a visible capsule [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn both \u003cem\u003eE. coli\u003c/em\u003e and KP, the protein RfaH plays a critical role in maintaining cell envelope integrity. Deletion of RfaH in \u003cem\u003eE. coli\u003c/em\u003e results in dramatic sensitivity to the detergent sodium dodecyl sulfate (SDS), mirroring the effect of a polar mutation within its target operon, which was responsible for lipopolysaccharide (LPS) biosynthesis [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This sensitivity can be alleviated by mutations in rho, a termination factor [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. KP exhibits a similar reliance on RfaH, with its deletion leading to reduced capsule production [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This parallels observations in \u003cem\u003eE. coli\u003c/em\u003e, suggesting a conserved role for RfaH in capsule biosynthesis due to the close resemblance of the corresponding gene clusters in both species [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMutations within genes encoding rfaH, in \u003cem\u003eK. pneumoniae\u003c/em\u003e, have also been linked to the development of phage resistance in this bacterium. RfaH plays an essential role in regulating and synthesizing both CPS and LPS, which are important components of the bacterial cell wall. Studies have consistently shown that mutations affecting genes involved in CPS and/or LPS synthesis contribute to phage resistance. This resistance mechanism arises from the loss of specific receptors on the bacterial surface due to the altered cell wall composition, hindering phage attachment and subsequent infection [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. These findings highlight the crucial contribution of RfaH to cell envelope stability and its intricate interplay with other regulatory factors like Rho in both \u003cem\u003eE. coli\u003c/em\u003e and KP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn \u003cem\u003eK. pneumoniae\u003c/em\u003e ATCC 700603, treatment with 100 \u0026micro;M Rifaximin led to a dramatic reduction in capsule production, exceeding 50% compared to untreated controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e To further affirm the effect of Rifaximin on KP outer capsule, 6 hours cultivated \u003cem\u003eK. pnuemoniae\u003c/em\u003e cells were negatively stained and visualized [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As observed, Rifaximin treated and untreated \u003cem\u003eK. pnuemoniae\u003c/em\u003e cells showed variation in bacterial outer capsule thickness. The untreated cells showed comparatively thicker outer capsule when compared to sub-MIC Rifaximin treated cells that showed smaller cells with relatively thin outer capsule \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This observation suggests a potential role for Rifaximin in inhibiting RfaH, a critical protein in the KP capsule biosynthesis pathway.\u003c/p\u003e \u003cp\u003eThe capsule, a polysaccharide-based extracellular structure, serves as a vital shield against diverse environmental assaults, including phagocytosis by the immune system, phage infection, and desiccation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Consequently, a substantial decrease in capsule content, as induced by Rifaximin, is likely to heighten the susceptibility of \u003cem\u003eK. pneumoniae\u003c/em\u003e cells to these external stresses, potentially culminating in cell death. Further investigation is warranted to elucidate the precise mechanism by which Rifaximin interacts with RfaH and to assess the functional impact of this capsule reduction on KP virulence and its overall adaptability within the host and environment [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe molecular docking between RfaH and Rifaximin revealed a binding affinity of -9.3 kcal/mol. An in-depth analysis of the interaction between Rifaximin and the RfaH binding pocket was conducted for all 9 docked conformations of the ligand. While Rifaximin displayed interactions with RfaH at multiple sites, its favored docking position exhibited the highest binding strength when compared to alternative positions. Literature studies have demonstrated that disrupting specific contact points in Eco RfaH, namely with the β\u0026rsquo;CH (Tyr54), ops DNA (Arg73), βGL (Thr66), and S10 (Ile146), impairs RfaH-dependent gene activation in \u003cem\u003eE. coli\u003c/em\u003e and the functional importance of these contact points extends to \u003cem\u003eK. pneumoniae\u003c/em\u003e also. As anticipated, disrupting interactions with β'CH, S10, and ops DNA, abolishes the activity of operons regulated under RfaH. This suggests that some RfaH interaction points are universally critical for both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e bacterial species [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Our docking results indicated that the rifaximin forms bonds with the Tyr54, Phe78, Arg80, and Asp147 residues of the RfaH protein. As Tyr54 which is a critical residue of β\u0026prime; clamp helices (CH) domain and thus for RfaH activity, hence disruption of this specific contact point by rifaximin will lead to RfaH inhibition. The binding configuration of Rifaximin with RfaH is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Rifaximin binds within the binding pocket cavity of RfaH, interacting with residues situated in the β\u0026prime; clamp helices (CH) domain, as depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC indicates a deep penetration of Rifaximin to the pocket of RfaH.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMD simulations\u003c/h2\u003e \u003cp\u003eThe MD simulation is employed to gain insights into the atomic-level dynamics of protein-ligand complexes [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Moreover, it aids in evaluating the flexibility of the docked complex in comparison to the native protein state [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In this investigation, MD simulation was applied to appraise the stability of protein-ligand docked complexes, specifically those involving RfaH and Rifaximin within a water model. Employing the CHARMM36 force field, we conducted a 100-nanosecond simulation for both the RfaH and RfaH-Rifaximin complex. Detailed information on MD simulation outcomes is provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVarious dynamic and structural parameters were examined and analyzed for a period of 100 ns for the RfaH-Rifaximin complex.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRMSD (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRMSF (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003eg (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSASA (nm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e#H-bonds\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRfaH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e114.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRfaH-Rifaximin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e114.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe protein undergoes structural dynamics upon binding to a ligand molecule. The calculation of root mean square deviation (RMSD) is a fundamental method employed to quantify the structural alterations in a protein following ligand binding. RMSD values for both the native state and the protein-ligand complex are charted across a 100 ns timeframe. The RfaH native structure exhibits slight fluctuations during this period. Notably, the time evolution of RMSD for the RfaH-Rifaximin complex displays some fluctuations after 60 ns, indicating that the system undergoes complexity and experiences minor instability during the simulation period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring MD simulation, the residual flexibility of a protein over duration is assessed through a root mean square fluctuation (RMSF) plot. We have generated RMSF plots for both the RfaH native state and the RfaH-Rifaximin complex (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Throughout the simulation, the RfaH-Rifaximin complex exhibits slightly elevated RMSF values. Despite this, both systems demonstrate nearly synchronized RMSF distributions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. This implies that the protein-ligand complex maintains stability post-binding. The RMSF analysis reveals minimal differences in RMSF between the two systems, signifying a stable complex.\u003c/p\u003e \u003cp\u003eIn MD simulation, the evaluation of the structural folding and conformational dynamics of a protein involves estimating the radius of gyration (\u003cem\u003eR\u003c/em\u003eg). The \u003cem\u003eR\u003c/em\u003eg is computed by determining the average distance of each atom from the center of mass of the protein molecule, employing the square of each atom's distance. Throughout the simulation, variations in the size and shape of the protein molecule are observed through the \u003cem\u003eR\u003c/em\u003eg, providing insights into the stability of the protein. Throughout a 100 ns duration, we computed the \u003cem\u003eR\u003c/em\u003eg values for both the RfaH native structure and the RfaH-Rifaximin complex \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. There is not any significant difference in the \u003cem\u003eR\u003c/em\u003eg values between both systems. By the 100 ns mark, the \u003cem\u003eR\u003c/em\u003eg values for both the free protein and the complex converge. This convergence suggests that RfaH, upon binding to Rifaximin, exhibits stable conformational dynamics and folding.\u003c/p\u003e \u003cp\u003eThe solvent-accessible surface area (SASA) indicates the portion of the protein's surface that is accessible to solvent molecules. Assessing the solvent-accessible surface area is a pivotal technique for evaluating interactions between a protein and a ligand. This measure is valuable in evaluating the stability of the protein-ligand complex and identifying potential binding sites. The analysis of SASA is widely used to understand the stability and folding properties of proteins and protein-ligand complexes. We have graphed the SASA values for both the RfaH native structure and the RfaH-Rifaximin complex throughout the entire simulation duration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e The mean SASA value for both systems remains constant, exhibiting no significant alteration. Throughout the simulation period, no notable distinctions are observed, and the values converge by 100 ns. This convergence implies a stable structural folding and dynamics of the protein upon binding to the ligand.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDynamics of hydrogen bonds\u003c/h2\u003e \u003cp\u003eThe stability of the protein-ligand complex relies on the establishment of hydrogen bonds [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Intra-molecular hydrogen bonds were calculated for both the RfaH native structure and RfaH after binding with Rifaximin, and the results were plotted over 100 ns duration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The plot generated indicates that there were no significant alterations observed in the hydrogen bonding interactions within the RfaH protein upon the formation of a complex with Rifaximin. The plot displays a constant number of hydrogen bonds for both systems. Intermolecular hydrogen bonds were also estimated to infer the stability of interactions of RfaH with Rifaximin. These interactions revealed the formation of up to six hydrogen bonds, with four consistently present bonds throughout the trajectory \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of secondary structures\u003c/h2\u003e \u003cp\u003eExamining the dynamics of the secondary structure content of protein is a means to understand its conformational behavior and folding mechanism [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. We calculated the alterations in the secondary structure for RfaH when bound to Rifaximin. The structural elements in the unbound RfaH exhibit nearly constant and equilibrated characteristics throughout the 100 ns simulation period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e However, a small decrease in the β-sheets and a slight increase in α-helix and content of RfaH can be seen upon compound binding \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The average number of residues engaged in secondary structure formation differs in the case of the RfaH-Rifaximin complex compared to free RfaH (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Despite this, there is no major change observed in the secondary structure of RfaH upon the binding of Rifaximin, indicating strong stability of the complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of Secondary Structures during MD simulation of RfaH native and RfaH-Rifaximin complex\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRfaH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRfaH-Rifaximin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-sheet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-bridge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBend\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTurn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-helix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003csub\u003e10\u003c/sub\u003e-helix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePCA and FEL analysis\u003c/h2\u003e \u003cp\u003eThe PCA is a crucial technique for assessing the collective motion of atoms in a protein-ligand complex [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. PCA is employed to analyze the conformational changes in both the native RfaH and the RfaH-Rifaximin complex, utilizing projections of Cα atoms to estimate the conformational dynamics of these systems. The plots illustrating these analyses are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA. The subspaces occupied by free RfaH closely align with those of the protein-ligand complexes. The native RfaH occupies one subspace, while the complex occupies two subspaces, signifying a reduction in stability within the complex. The protein-ligand systems show some variability concerning the free state of the protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAssessing the protein folding mechanism involves the utilization of Free Energy Landscape (FEL) analysis. This analysis is employed to determine global and local minima points within the energy landscape of a protein. The FEL plots for both the unbound RfaH and the RfaH-Rifaximin complex are depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB \u003cb\u003eand C\u003c/b\u003e. Regions in deep blue color within the plots represent low-energy states, closely associated with the native states. The free state of the protein showed one large basin. In the case of RfaH-Rifaximin three distinct basins are formed. It suggests that the global minimum of free RfaH is slightly disturbed by the binding of Rifaximin. In summary, the FEL analysis suggests that the interaction of Rifaximin with RfaH does not induce protein unfolding throughout the 100 ns timeframe.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe alarming rise of antimicrobial resistance (AMR) in KP necessitates a multi-pronged approach, including judicious antibiotic use, enhanced infection control, and continual exploration of novel therapeutic strategies. Repurposing existing drugs, for example, the rapid FDA approval of Remdesivir for COVID-19 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], presents a promising avenue for combating KP infections. Rifaximin shows broad-spectrum bactericidal activity against various enteric pathogens. Encompassing both gram-positive and negative, aerobic and anaerobic bacteria, this efficacy extends to a diverse range of enterotoxic and pathogenic species. A comprehensive microbiological survey conducted revealed a minimum inhibitory concentration (MIC90) ranging from 4 to 64 \u0026micro;g/ml for enteric pathogens like \u003cem\u003eEscherichia coli, Salmonella, Shigella, Campylobacter, Plesiomonas, and Aeromonas\u003c/em\u003e, isolated across three continents [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. These findings align with observations from other studies, solidifying the consistent susceptibility patterns of these bacteria towards Rifaximin [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Notably, Rifaximin readily achieves concentrations exceeding 8,000 \u0026micro;g/g within human feces, effectively surpassing the MIC90 values and ensuring potent action against these enteric pathogens within the gastrointestinal tract [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile Rifaximin boasts high gut concentrations and a broad spectrum of antibacterial activity, it exhibits a surprising lack of significant disruption to the intestinal microbiota. A two-week course of Rifaximin in human subjects resulted in just a 1 log reduction in intestinal coliforms per gram of stool, highlighting its minimal impact on the overall gut microbial population [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Due to its minimal systemic absorption, Rifaximin also has a favorable safety profile with a low occurrence of adverse events. In clinical trials involving over 1000 participants, those receiving Rifaximin reported side effects at frequencies comparable to or even lower than those observed in groups given placebo, ciprofloxacin, or TMP-SMX, further solidifying its reputation as a well-tolerated medication [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. These characteristics make Rifaximin a valuable therapeutic option offering a distinct advantage over traditional antibiotics, which often cause substantial collateral damage to the beneficial gut microbiome and mitigating potential side effects.\u003c/p\u003e \u003cp\u003eRifamixin is a structural analog of Rifampicin [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. We have done \u003cem\u003ein-silico\u003c/em\u003e studies showing both Rifampicin and Rifamixin share the same binding pocket on DNA-dependent RNA polymerase (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A recent study has suggested that Rifampicin binds to the beta subunit (\u003cem\u003erpoB\u003c/em\u003e) of DNA-dependent RNA polymerase in hypermucoviscous strain of \u003cem\u003eKlebseilla pneumoniae\u003c/em\u003e that reduces its mucoviscosity. However, there is a persistent mystery regarding its mechanism of action as the authors do not believe that sterically blocked Rifampicin-bound RNA polymerase plays an active role in the anti-mucoviscouscos mechanism \u003cem\u003ei.e.\u003c/em\u003e reducing capsule production [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Mutations in the \u003cem\u003erpoB\u003c/em\u003e of RNA polymerase, leads to alteration in the phenotypic traits of bacterial cells by affecting transcription elongation and Rho-dependent and Rho-independent termination [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In \u003cem\u003eE.coli\u003c/em\u003e rho mutants showed changes in the expression of \u003cem\u003erfaH\u003c/em\u003e genes forming altered outer membrane or capsule production [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The overall decrease in transcriptional activity leads to change in the gene expression pattern by inducing local and global cellular responses. The sub-MIC levels of antibiotics irrespective of their mode of action show transcriptional modulatory effects [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. In the case of \u003cem\u003eSalmonella typhimurium\u003c/em\u003e, many antimicrobials modulate the transcription of subset genes. These antimicrobials are of different structures and have different modes of action [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA detailed study involving Rifampicin and \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e transcriptional modulations has been done. The study reveals that many subsets of genes are being down-regulated as well as up-regulated as an effect of sub-MIC levels of Rifampicin. In spite of an in-depth analysis of the authors regarding the divergently affected promoter the precise mechanism remains unclear for their observations [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Based on these studies, we can speculate that as seen in the \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e and its global transcriptional changes may happen in \u003cem\u003eKlebseilla pnuemoniae\u003c/em\u003e when subjected to sub-MIC levels of Rifamixin leading to changes in the gene expression of RfaH and hence capsule production.\u003c/p\u003e \u003cp\u003eOur study provides a foundation for the potential repurposing of rifaximin in the fight against KP infections. While its limited absorption within the gut presents a potential hurdle in future in-vivo studies, our in-vitro findings demonstrate its antibacterial activity against KP through its interaction with the RfaH protein. This crucial discovery opens avenues for developing novel RfaH inhibitors with improved gut absorption properties based on the structural framework of Rifaximin. By synergistically integrating insights from disease pathophysiology, drug pharmacology, and computational analysis, we can prioritize promising drug candidates for KP infections. This interdisciplinary approach holds immense potential for accelerating the development of effective KP therapies, ultimately improving patient outcomes and mitigating the healthcare burden.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn light of the rising threat posed by multidrug-resistant KP, repurposing existing FDA-approved drugs emerges as a promising strategy. This study explores the potential of rifaximin, a non-absorbable oral antibiotic currently used for irritable bowel syndrome (IBS) [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], as a novel therapeutic against KP infections. Our exploration of the drug repurposing potential of rifaximin as a targeted therapeutic against KP, leveraging the inhibition of the transcription anti-termination protein RfaH, reveals a promising avenue for novel antimicrobial development. The remarkable antimicrobial activity demonstrated by Rifaximin, coupled with its significant reduction in capsule production in KP, suggests its efficacy in obstructing the crucial activities of RfaH. A strong binding affinity of Rifaximin to RfaH evidenced by fluorescence studies, and the stable MD simulation data further elucidate the intricate and sustained interaction between rifaximin and RfaH. These findings collectively emphasize the multifaceted potential of rifaximin as a repurposed antibiotic that can disrupt key virulence mechanisms in KP. However, moving forward, a rigorous exploration and validation of rifaximin's potential in clinical settings are imperative to implicate its full therapeutic capabilities and revolutionize novel approaches to combat bacterial infections.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eKP: \u003cem\u003eKlebsiella pneumoniae,\u003c/em\u003e MDR-KP: Multidrug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eR\u003c/em\u003e\u003csub\u003eg\u003c/sub\u003e: Radius of gyration, MIC: Minimum Inhibitory Concentration, MBC: Minimum Bactericidal Concentration, MD: Molecular Dynamics, RMSD: Root Mean Square Deviation, RMSF: Root Mean Square Fluctuation, SASA: Solvent Accessible Surface Area\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDisclosure \u0026nbsp;statement\u0026nbsp;No \u0026nbsp; potential \u0026nbsp;conflict \u0026nbsp;of \u0026nbsp; interest \u0026nbsp;was \u0026nbsp;reported \u0026nbsp; by \u0026nbsp;the \u0026nbsp;author(s).\u003c/p\u003e\n\u003cp\u003eData Availability Statement:\u0026nbsp;The information supporting this study is available in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work is funded by the Indian Council of Medical Research (Grant No. ECD/adoc/2/2021-22).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eAA thanks to Indian Council of Medical Research-Department of Health Research (File No. R.12014/06/2022-HR) for financial support. MIH thanks the Indian Council of Medical Research for financial support (Grant No. ECD/adoc/2/2021-22).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCredit author statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnam Ashraf\u003c/strong\u003e: Conceptualization, Investigation, Methodology, Writing- Original draft preparation,\u0026nbsp;\u003cstrong\u003eArunabh Chaudhary\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Data curation, Graphics,\u0026nbsp;Data validation;\u0026nbsp;\u003cstrong\u003eMohammad Ali Khan:\u003c/strong\u003e Methodology,\u0026nbsp;Data validation, Writing-review, and editing;\u0026nbsp;\u003cstrong\u003eSaba Noor:\u003c/strong\u003e Graphics,\u0026nbsp;Data validation, Writing-review, and editing; \u003cstrong\u003eAsimul Islam:\u003c/strong\u003e Data curation, Graphics,\u0026nbsp;Data validation;\u0026nbsp;\u003cstrong\u003eMd. Imtaiyaz Hassan:\u0026nbsp;\u003c/strong\u003eConceptualization, Investigation, Supervision, Review and editing, and project administration.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKarampatakis T, Tsergouli K, Behzadi P (2023) Carbapenem-resistant Klebsiella pneumoniae: Virulence factors, molecular epidemiology and latest updates in treatment options. Antibiotics 12:234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRusso A, Fusco P, Morrone HL, Trecarichi EM, Torti C (2023) New advances in management and treatment of multidrug-resistant Klebsiella pneumoniae. Expert Rev Anti-infective Therapy 21:41\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpadar A, Phelan J, Elias R, Modesto A, Caneiras C, Marques C, Lito L, Pinto M, Cavaco-Silva P, Ferreira H (2022) Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistance. Sci Rep 12:13791\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHallal Ferreira Raro O, Nordmann P, Dominguez Pino M, Findlay J, Poirel L (2023) Emergence of carbapenemase-producing hypervirulent Klebsiella pneumoniae in Switzerland. Antimicrob Agents Chemother 67:e01424\u0026ndash;e01422\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C (2019) Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discovery 18:41\u0026ndash;58\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalaramnavar VM, Ahmad K, Saeed M, Ahmad I, Kamal M, Jawed T (2020) Pharmacophore-based approaches in the rational repurposing technique for FDA approved drugs targeting SARS-CoV-2 M pro. RSC Adv 10:40264\u0026ndash;40275\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan Baig M, Ahmad K, Roy S, Mohammad Ashraf J, Adil M, Haris Siddiqui M, Khan S, Kamal A, Provazn\u0026iacute;k M, I., and, Choi I (2016) Computer aided drug design: success and limitations. Curr Pharm Design 22:572\u0026ndash;581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou JX, Torres VE (2023) Drug repurposing in ADPKD. Kidney International\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarha MA, Brown ED (2019) Drug repurposing for antimicrobial discovery. 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Nat Reviews Gastroenterol Hepatol 8:121\u0026ndash;121\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rifaximin, Anti-termination Protein RfaH, MD simulation, Fluorescence studies, Anti-biotic resistance","lastPublishedDoi":"10.21203/rs.3.rs-4724428/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4724428/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnti-termination protein RfaH plays a crucial role in promoting virulence across various Gram-negative pathogens, including \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (KP). RfaH directly interacts with RNA-polymerase and ribosomes, which in turn facilitates the activation of operons associated with capsule, cell wall, and pilus biosynthesis. This study aimed to investigate the repurposing potential of rifaximin, a well-established antibiotic, against KP by strategically targeting RfaH, a pivotal anti-terminator protein in transcription. Fluorescence studies observed an excellent binding affinity between rifaximin and RfaH (\u003cem\u003eK\u003c/em\u003ea\u0026thinsp;=\u0026thinsp;7.38 x 10\u003csup\u003e6\u003c/sup\u003eM\u003csup\u003e\u0026minus;1\u003c/sup\u003e). Intriguingly, rifaximin treatment causes a significant reduction in capsule production in KP when compared to untreated controls, elucidating its inhibitory influence on RfaH activity. The minimum inhibitory concentration for Rifaximin was calculated as 100\u0026micro;M and a minimum bactericidal concentration of 200\u0026micro;M against KP (ATCC 700603 strain). Docking and MD simulation studies provided detailed atomic insights into the Rifaximin binding to RfaH and structural dynamics of the RfaH-Rifaximin complex. These multifaceted findings collectively investigated the potential of rifaximin as a repurposed antibiotic against KP. Finally, a strong interaction of RfaH with rifaximin and subsequent inhibition of the growth of KP provides a novel avenue for antimicrobial development for addressing the persistent global challenge of antibiotic-resistant infections.\u003c/p\u003e","manuscriptTitle":"Repurposing Rifaximin against Klebsiella pneumoniae via Targeting of Transcription Anti-termination Protein RfaH for Novel Antimicrobial Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 13:08:39","doi":"10.21203/rs.3.rs-4724428/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a65f747c-15ad-4ad1-a6d9-230d0cc28863","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T20:03:48+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-07 13:08:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4724428","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4724428","identity":"rs-4724428","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}