Repurposing of FDA approved drugs against uropathogenic Escherichia coli: In silico, in vitro, and in vivo analysis.

Urinary tract infections (UTIs) are a serious health concern worldwide. Treatment of UTIs is becoming a challenge as uropathogenic Escherichia coli (UPEC), which is the most common etiological agent, has developed resistance to the main classes of antibiotics. Small molecules that interfere with metabolic processes rather than growth are attractive alternatives to conventional antibiotics. Repurposing of already known drugs for treating infectious diseases could be an attractive avenue for finding novel therapeutics against infections caused by UPEC. Virtual screenings enable the rapid and economical identification of target ligands from large libraries of compounds, reducing the cost and time of traditional drug discovery. Moreover, the drugs that have been approved by the FDA have low cytotoxicity and good pharmacological characteristics. In this work, we targeted the HisC enzyme of the histidine biosynthetic pathway as enzymes of this pathway are absent in humans. We screened the library of FDA-approved drugs against HisC via molecular docking, and four hits (Docetaxel, Suramin, Digitoxin, and Nystatin) showing the highest binding energy were selected. These were further tested for antibacterial activity, which was observed only for Docetaxel (MIC value of 640 µg/ml); therefore, Docetaxel was further tested for its efficacy in vivo in murine catheter UTI model and antibiofilm activity using crystal violet staining and scanning electron microscopy. Docetaxel inhibited biofilm formation and reduced the bacterial load in urine, kidney, and bladder. Docking studies revealed that Docetaxel acts by blocking the binding site of HisC to the native substrate by competitive inhibition. Docetaxel may be a potential new inhibitor for UPEC with antibacterial and antibiofilm capability.

Identification of novel non-homologous drug targets against Acinetobacter baumannii using subtractive genomics and comparative metabolic pathway analysis.

Lack of effective antibiotics and the development of multidrug resistance in clinical isolates of nosocomial pathogen Acinetobacter baumanni has necessitated the identification of novel drug targets. The study is divided into three phases, in phase I, four different sets of proteins were subjected to a chokepoint, plasmid, resistance genes, and virulence factors analysis. After phase 1 analysis we obtained two hundred twenty-two proteins which were analyzed further in the phase II for essentiality and homology. Fifty-eight proteins identified as target candidates were studied for qualitative characteristics. Among them, 32 were identified as cytoplasmic membrane, 17 as cytoplasmic, one as periplasmic, one as outer membrane, two as extracellular, and location of 5 was not known. Druggability analysis revealed that 18 proteins were druggable, and 40 were novel. Drug targets obtained in the present study can be utilized for the identification of novel antimicrobials for the treatment of infections caused by multidrug-resistant A. baumannii. Predicted drug targets can be evaluated for their binding affinity by molecular docking studies and thus accelerating the process of drug discovery.

Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxo-dodecanoyl)-L-homoserine lactone triggers mitochondrial dysfunction and apoptosis in neutrophils through calcium signaling.

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes the quorum-sensing (QS) process to regulate the production of different virulence factors and biofilm. N-3-oxo-dodecanoyl-L-homoserine lactone (C12) is a key QS molecule of P. aeruginosa which interacts with the mammalian immune cells and modulates their function. Here, we investigated the molecular mechanism of C12-induced apoptosis in neutrophils. Our data show that C12 causes apoptosis in neutrophils through an elevation in cytosolic and mitochondrial Ca2+ levels. Besides, C12 induces phosphatidylserine (PS) exposure, mitochondrial membrane potential (MMP) depolarization, mitochondrial permeability transition pore (MPTP) formation and mitochondrial reactive oxygen species (mROS) generation. C12-induced rise in intracellular Ca2+ level is majorly contributed by endoplasmic reticulum store through the activation of inositol 1, 4, 5-triphosphate receptor. Intracellular calcium chelation inhibited C12-induced mitochondrial dysfunction and apoptosis. Further, inhibition of mitochondrial Ca2+ uniporter by ruthenium red or Ru360 abrogated C12-induced mitochondrial Ca2+ uptake, MMP loss, MPTP opening, mROS production, and PS exposure. These mechanistic insights are expected to provide a better understanding of the role of C12 in P. aeruginosa pathogenesis.

Pseudomonas aeruginosa quorum sensing molecule N-3-oxo-dodecanoyl-l-homoserine lactone activates human platelets through intracellular calcium-mediated ROS generation.

Pseudomonas aeruginosa, an opportunistic pathogen release N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C12HSL) and N-butyryl-l-homoserine lactone (C4-HSL) quorum sensing (QS) molecules to regulate various virulence factors responsible for infection in the host. 3-oxo-C12 HSL not only regulates the bacterial gene expression but also modulates the host cell system. Thus, it is pertinent to evaluate the effect of these QS molecules on blood platelets which is responsible for the maintenance of hemostasis and thrombus formation. Here, in the present study, we showed that 3-oxo-C12 HSL activates platelets in a dose-dependent manner and induces intracellular calcium-mediated reactive oxygen species (ROS) release, whereas no such effect was observed with C4-HSL. 3-oxo-C12 HSL stimulated ROS release was mediated by NADPH oxidase. Results confirmed the involvement of phospholipase C (PLC) and IP3 receptor behind intracellular calcium-mediated ROS generation. The impact of 3-oxo-C12 HSL on platelet activation suggests that it could interfere and alter the normal function of platelet in individuals infected with P. aeruginosa.

Pseudomonas aeruginosa auto inducer3-oxo-C12-HSL exerts bacteriostatic effect and inhibits Staphylococcus epidermidis biofilm.

Pseudomonas aeruginosa has evolved the 3-oxo-C12-HSL and C4-HSL based quorum sensing system which is responsible for the regulation of various virulence factors and helps to dominates over other bacterial species. Staphylococcus epidermidis has frequently been reported with P. aeruginosa while the role of C4-HSL and 3-oxo-C12-HSL on the S. epidermidis had widely been unexplored, and as per our knowledge, this is the first report on the impact of C4-HSL and 3-oxo-C12-HSL overS. epidermidis growth and biofilm. We found that among the two AHL molecules; only 3-oxo-C12-HSL was able to exert a significant effect in all the experiments including growth and biofilm of S. epidermidis. 3-oxo-C12-HSL at 100 µM and 200 µM concentrations were able to initiate the apparent transient type of planktonic growth inhibition in S. epidermidis. Microscopic analysis and biofilm quantification assay showed the inhibitory effect of 3-oxo-C12-HSL against S. epidermidis biofilm, initial attachment, and EPS production. The study concludes that P. aeruginosa associated 3-oxo-C12-HSL exerts the inhibitory effect on S. epidermidis growth and biofilms and thus it may also help Pseudomonasto dominate under the co-infection conditions.

Structure based virtual screening for identification of potential quorum sensing inhibitors against LasR master regulator in Pseudomonas aeruginosa.

Inter and intracellular communication in bacteria, which is known as quorum sensing (QS), is mediated by small diffusible signaling molecules known as autoinducers. QS regulates various virulence factors responsible for pathogenesis. Increasing resistance of microorganisms against traditional antibiotics has turned the focus towards the QS as it exerts less selective pressure preventing development of resistance among microorganisms. LasR, a transcription factor that controls QS in Pseudomonas aeruginosa, is an attractive therapeutic target for inhibitors. This study aimed to screen natural compounds as potential inhibitors of LasR. About 2603 compounds from ZINC database were virtually screened against the structure of LasR. Then after qualifying compounds were filtered on the parameters of Lipinski's rule and ADME. Six novel potential QS inhibiting compounds were selected on the basis of binding energy. Structures of LasR-ligand complexes were analysed to have insight of binding between inhibitors and target. It is pertinent to mention here that all the molecules are structurally different from 3-oxo-C12HSL,a native autoinducer of LasR, that play key role in formation of LasR dimer which is an active form of the protein to facilitate QS.

Identification of novel inhibitors against Escherichia coli utilizing HisC as a target from histidine biosynthesis pathway.

Escherichia coli is a gram-negative bacterial pathogen that poses a significant challenge both clinically and epidemiologically. Large numbers of multi-drug resistant E. coli have emerged in the last decade, because of the selection pressure generated by the inadequate use of antibiotics. Although research to combat antibiotic resistance has been going on extensively but still lags in the rate of development of newer antibiotics. Therefore, newer approaches are required to speed up the rate of discovery of antibiotics. Computational methods for screening of inhibitors have made a significant contribution to the discovery of novel antimicrobials. The present study utilized histidinol-phospho aminotransferase (HisC) as a target. HisC is an enzyme that plays a crucial role in the biosynthesis of histidine and its absence in mammals makes it an attractive drug target. A ZINC library of 5000 natural compounds was screened against HisC (PDB ID: 1FG7) using PyRx and the first 500 hits were selected for secondary screening after sorting the result on the basis of binding score. Fifteen compounds passed the secondary filter ADME and out of these five passed toxicity filters; the best among five hits was selected on the basis of its binding score and inhibition constants. Further, molecular dynamics simulations and free binding were computed of selected five compounds and two natural compounds ZINC402598829 and ZINC31157928 complexed with HisC were found as highly stable. Overall, our results indicated that these natural sources could be used as potential HisC inhibitors.Communicated by Ramaswamy H. Sarma.

cis-DA-dependent dispersion by Pseudomonas aeruginosa biofilm and identification of cis-DA-sensory protein DspS.

IMPORTANCE: Dispersion is an essential stage of the biofilm life cycle resulting in the release of bacteria from a biofilm into the surrounding environment. Dispersion contributes to bacterial survival by relieving overcrowding within a biofilm and allowing dissemination of cells into new habitats for colonization. Thus, dispersion can contribute to biofilm survival as well as disease progression and transmission. Cells dispersed from a biofilm rapidly lose their recalcitrant antimicrobial-tolerant biofilm phenotype and transition to a state that is susceptible to antibiotics. However, much of what is known about this biofilm developmental stage has been inferred from exogenously induced dispersion. Our findings provide the first evidence that native dispersion is coincident with reduced cyclic dimeric guanosine monophosphate levels, while also relying on at least some of the same factors that are central to the environmentally induced dispersion response, namely, BdlA, DipA, RbdA, and AmrZ. Additionally, we demonstrate for the first time that cis-DA signaling to induce dispersion is attributed to the two-component sensor/response regulator DspS, a homolog of the DSF sensor RpfC. Our findings also provide a path toward manipulating the native dispersion response as a novel and highly promising therapeutic intervention.

In Vitro and In Vivo Studies of Heraclenol as a Novel Bacterial Histidine Biosynthesis Inhibitor against Invasive and Biofilm-Forming Uropathogenic Escherichia coli.

Globally, urinary tract infections (UTIs) are one of the most frequent bacterial infections. Uropathogenic Escherichia coli (UPEC) are the predominant etiological agents causing community and healthcare-associated UTIs. Biofilm formation is an important pathogenetic mechanism of UPEC responsible for chronic and recurrent infections. The development of high levels of antimicrobial resistance (AMR) among UPEC has complicated therapeutic management. Newer antimicrobial agents are needed to tackle the increasing trend of AMR and inhibit biofilms. Heraclenol is a natural furocoumarin compound that inhibits histidine biosynthesis selectively. In this study, for the first time, we have demonstrated the antimicrobial and antibiofilm activity of heraclenol against UPEC. The drug reduced the bacterial load in the murine catheter UTI model by ≥4 logs. The drug effectively reduced bacterial loads in kidney, bladder, and urine samples. On histopathological examination, heraclenol treatment showed a reversal of inflammatory changes in the bladder and kidney tissues. It reduced the biofilm formation by 70%. The MIC value of heraclenol was observed to be high (1024 µg/mL), though the drug at MIC concentration did not have significant cytotoxicity on the Vero cell line. Further molecular docking revealed that heraclenol binds to the active site of the HisC, thereby preventing its activation by native substrate, which might be responsible for its antibacterial and antibiofilm activity. Since the high MIC of heraclenol is not achievable clinically in human tissues, further chemical modifications will be required to lower the drug's MIC value and increase its potency. Alternatively, its synergistic action with other antimicrobials may also be studied.

The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms.

Dispersion is an active process exhibited by Pseudomonas aeruginosa during the late stages of biofilm development or in response to various cues, including nitric oxide and glutamate. Upon cue sensing, biofilm cells employ enzymes that actively degrade the extracellular matrix, thereby allowing individual cells to become liberated. While the mechanism by which P. aeruginosa senses and relays dispersion cues has been characterized, little is known about how dispersion cue sensing mechanisms result in matrix degradation. Considering that the alginate and motility regulator AmrZ has been reported to regulate genes that play a role in dispersion, including those affecting virulence, c-di-GMP levels, Pel and Psl abundance, and motility, we asked whether AmrZ contributes to the regulation of dispersion. amrZ was found to be significantly increased in transcript abundance under dispersion-inducing conditions, with the inactivation of amrZ impairing dispersion by P. aeruginosa biofilms in response to glutamate and nitric oxide. While the overexpression of genes encoding matrix-degrading enzymes pelA, pslG, and/or endA resulted in the dispersion of wild-type biofilms, similar conditions failed to disperse biofilms formed by dtamrZ. Likewise, the inactivation of amrZ abrogated the hyperdispersive phenotype of PAO1/pJN-bdlA_G31A biofilms, with dtamrZ-impaired dispersion being independent of the expression, production, and activation of BdlA. Instead, dispersion was found to require the AmrZ-target genes napB and PA1891. Our findings indicate that AmrZ is essential for the regulation of dispersion by P. aeruginosa biofilms, functions downstream of BdlA postdispersion cue sensing, and regulates the expression of genes contributing to biofilm matrix degradation as well as napB and PA1891. IMPORTANCE In P. aeruginosa, biofilm dispersion has been well-characterized with respect to dispersion cue perception, matrix degradation, and the consequences of dispersion. While the intracellular signaling molecule c-di-GMP has been linked to many of the phenotypic changes ascribed to dispersion, including the modulation of motility and matrix production, little is known about the regulatory mechanisms leading to matrix degradation and cells actively leaving the biofilm. In this study, we report for the first time an essential role of the transcriptional regulator AmrZ and two AmrZ-dependent genes, napB, and PA1891, in the dispersion response, thereby linking dispersion cue sensing via BdlA to the regulation of matrix degradation and to the ultimate liberation of bacterial cells from the biofilm.

Identification and functional annotation of hypothetical proteins of uropathogenic Escherichia coli strain CFT073 towards designing antimicrobial drug targets.

Urinary tract infections are a serious health concern worldwide, especially in developing countries. Escherichia coli strain CFT073 is a highly virulent pathogenic bacterial strain. CFT073 proteome contains 4897 proteins, out of which 992 have been classified as hypothetical proteins. Identification and characterization of hypothetical proteins can aid in the selection of targets for drug design. In this study, we studied the hypothetical proteins from the UPEC strain CFT073 using various computational tools. By NCBI-CDD, 376 protein sequences showed conserved domains. Based on the functional motifs in their primary sequences, we classified these 376 hypothetical proteins into 7 functional categories. Further KEGG database was used to find the roles of these hypothetical proteins in several pathways. Protein interaction network analysis of hypothetical proteins identified 53 proteins as highly interacting metabolic proteins. Virulence factor analysis of the proteins identified 8 proteins as virulent. We conducted a non-homology search for the identified proteins of UPEC in the available human proteome. We observed that 35 proteins are non-homologous to humans and hence could be selected for drug designing targets. Qualitative characterization of the selected 35 non-homologous hypothetical proteins including essentiality analysis and evaluation of druggability by similarity search against drug bank database was performed. Out of these 35 proteins, three-dimensional structures of six proteins (NP_752562.1, NP_756345.1, NP_754893.1, NP_756600.2, NP_755264.1 and NP_752994.1) could be successfully modelled. These new annotations can help to better understand disease mechanisms at the molecular level, as well as provide new targets for drug development against the UPEC strain CFT073.Communicated by Ramaswamy H. Sarma.

Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E. coli in India.

Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that causes acute and chronic diarrhea in developed and industrialized nations in children. EAEC epidemiology and the importance of atypical EAEC (aEAEC) isolation in childhood diarrhea are not well documented in the Indian setting. A comparative analysis was undertaken to evaluate virulence, phylogeny, and antibiotic sensitivity among typical tEAEC versus aEAEC. A total of 171 EAEC isolates were extracted from a broad surveillance sample of diarrheal (N = 1210) and healthy children (N = 550) across North India. Polymerase chain reaction (PCR) for the aggR gene (master regulator gene) was conducted to differentiate tEAEC and aEAEC. For 21 virulence genes, we used multiplex PCR to classify possible virulence factors among these strains. Phylogenetic classes were identified by a multiplex PCR for chuA, yjaA, and a cryptic DNA fragment, TspE4C2. Antibiotic susceptibility was conducted by the disc diffusion method as per CLSI guidelines. EAEC was associated with moderate to severe diarrhea in children. The prevalence of EAEC infection (11.4%) was higher than any other DEC group (p = 0.002). tEAEC occurrence in the diarrheal group was higher than in the control group (p = 0.0001). tEAEC strain harbored more virulence genes than aEAEC. astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination was present in more than 50% of strains. Also, 75.8% of EAEC strains were multidrug-resistant (MDR). Phylogroup D (43.9%) and B1 (39.4%) were most prevalent in the diarrheal and control group, respectively. Genetic analysis revealed EAEC variability; the comparison of tEAEC and aEAEC allowed us to better understand the EAEC virulence repertoire. Further microbiological and epidemiological research is required to examine the pathogenicity of not only typical but also atypical EAEC.

Designing quorum sensing inhibitors of Pseudomonas aeruginosa utilizing FabI: an enzymic drug target from fatty acid synthesis pathway.

Pseudomonas aeruginosa infections are a leading cause of death in patients suffering from respiratory diseases. The multidrug-resistant nature of Pseudomonas is potentiated by a process known as quorum sensing. The aim of this study was to reveal new inhibitors of a well-validated but quite unexplored target, enoyl-ACP reductase, which contributes acyl chain lengths of N-acyl homoserine lactones that are major signaling molecules in gram-negative bacteria. In the present study, the crystal structure of FabI (PDB, ID 4NR0) was used for the structure-based identification of quorum sensing inhibitors of Pseudomonas aeruginosa. Active site residues of FabI were identified from the complex of FabI with triclosan and these active site residues were further used to screen for potential inhibitors from natural database. Three-dimensional structures of the 75 natural compounds were retrieved from the ZINC database and screened using PyRX software against FabI. Thirty-eight molecules from the initial screening were sorted on the basis of binding energy, using the known inhibitor triclosan as a standard. These molecules were subjected to various secondary filters, such as Lipinski's Rule of Five, ADME, and toxicity. Finally, eight lead-like molecules were obtained after their evaluation for drug-like characteristics. The present study will open a new window for designing QS inhibitors against P. aeruginosa.

Exploring the impact of parthenolide as anti-quorum sensing and anti-biofilm agent against Pseudomonas aeruginosa.

AIMS: Pseudomonas aeruginosa is a well-known pathogen responsible for various infections due to its capability to develop biofilm and various virulent phenotypes that are regulated by quorum sensing. Pathogenesis of the bacteria may be halted by interfering with the signaling molecules and the quorum sensing receptors. Therefore, the present study explores the potential of parthenolide, a sesquiterpene lactone of feverfew plant, as a promising candidate against P. aeruginosa PAO1 associated virulence factors and biofilm. MAIN METHODS: Effect of parthenolide on virulence and biofilm formation of P. aeruginosa was studied using standard protocols. Mechanism of action was studied using Real-time PCR as well as molecular docking studies. KEY FINDINGS: Significant decrease in virulence factors and biofilm formation was observed when treated with the sub-MIC concentration (1 mM) of parthenolide. Gene expression studies showed the down-regulation of autoinducer synthase (lasI, rhlI) as well as their receptors (lasR and rhlR) with remarked repression of lasR by 57% compared to the control. Biofilm-associated fluorescent microscopic studies after staining with FITC-ConA and propidium iodide showed reduced extracellular polymeric substance (EPS) production and killing of bacterial cells after treatment with parthenolide. SIGNIFICANCE: The study is important as it reports for the first time the potential of parthenolide as an anti-quorum and anti-biofilm agent. This study will be helpful in designing of new quorum sensing inhibitors that help in the eradication of infections and can be given in combination with the antibiotics.

Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa.

Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation.