Moonlighting proteins: beacon of hope in era of drug resistance in bacteria.

Moonlighting proteins (MLPs) are ubiquitous and provide a unique advantage to bacteria performing multiple functions using the same genomic content. Targeting MLPs can be considered as a futuristic approach in fighting drug resistance problem. This review follows the MLP trail from its inception to the present-day state, describing a few bacterial MLPs, viz., glyceraldehyde 3'-phosphate dehydrogenase, phosphoglucose isomerase glutamate racemase (GR), and DNA gyrase. Here, we carve out that targeting MLPs are the beacon of hope in an era of increasing drug resistance in bacteria. Evolutionary stability, structure-functional relationships, protein diversity, possible drug targets, and identification of new drugs against bacterial MLP are given due consideration. Before the final curtain calls, we provide a comprehensive list of small molecules that inhibit the biochemical activity of MLPs, which can aid the development of novel molecules to target MLPs for therapeutic applications.

Bactericidal activity of esculetin is associated with impaired cell wall synthesis by targeting glutamate racemase of Neisseria gonorrhoeae.

Neisseria gonorrhoeae (NG), the causative organism of gonorrhea, has been classified by the World Health Organization as 'Priority' two organism owing to its increased resistance to antibiotics and even failure of recommended dual therapy with ceftriaxone and azithromycin. As a result, the general and reproductive health of infected individuals is severely compromised. The imminent public health catastrophe of antimicrobial-resistant gonococci cannot be understated, as t he of severe complications and sequelae of infection are not only increasing but their treatment has also become more expensive. Tenacious attempts are underway to discover novel drug targets as well as new drugs to fight against NG. Therefore, a considerable number of phytochemicals have been tested for their remedial intercession via targeting bacterial proteins. The MurI gene encodes for an enzyme called glutamate racemase (MurI) that is primarily involved in peptidoglycan (PG) biosynthesis and is specific to the bacterial kingdom and hence can be exploited as a potential drug target for the treatment of bacterial diseases. Accordingly, diverse families of phytochemicals were screened in silico for their binding affinity with N. Gonorrhoeae MurI (NG-MurI) protein. Esculetin, one of the shortlisted compounds, was evaluated for its functional, structural, and anti-bacterial activity. Treatment with esculetin resulted in growth inhibition, cell wall damage, and altered permeability as revealed by fluorescence and electron microscopy. Furthermore, esculetin inhibited the racemization activity of recombinant, purified NG-MurI protein, one of the enzymes required for peptidoglycan biosynthesis. Our results suggest that esculetin could be further explored as a lead compound for developing new drug molecules against multidrug-resistant strains.

Identification of potential therapeutic targets in Neisseria gonorrhoeae by an in-silico approach.

Neisseria gonorrhoeae is a gram negative diplococcus bacterium and the causative agent of the sexually transmitted disease Gonorrhea. It has been recently given the status of "superbug" by World Health Organization because of the increasing antibiotic resistance and unavailability of a viable vaccine candidate. Over recent years, there have been increasing reports about the use of subtractive genomics to identify potential drug and vaccine targets. Our study utilizes codon biasing, a tool to identify the essential genes, in N. gonorrhoeae that could be utilized as novel therapeutic targets for drug or vaccine development. Through the screening of 2350 total genes, we present a list of 29 such drug candidate genes based on codon adaptation. Through the data-mining with BLAST2GO and InterProScan databases, we could predict the function of these 29 genes. These genes are involved in pivotal cellular functions like DNA replication, energy synthesis and metabolites production. This study also shortlists the essential genes of N. gonorrhoeae that could be used to target Neisseria. We identified a molecule/drug which can be used to target essential protein DapD (succinyltransferase).

Ethambutol targets the glutamate racemase of Mycobacterium tuberculosis-an enzyme involved in peptidoglycan biosynthesis.

Increasing drug resistance in pathogens including Mycobacterium tuberculosis (MTB) has been ascribed to mutations in the known target genes. However, many of these drugs have multiple targets; some of which have not been identified so far. Understanding the mechanism of action of these drugs holds a great promise in better management of disease especially by drug-resistant strains. In this study, we report glutamate racemase (MurI), a crucial enzyme of phase I peptidoglycan (PG) biosynthesis pathway of MTB, as an additional target of ethambutol (EMB). The effect on EMB on the MurI protein at structural and functional level was studied using different spectroscopic, biochemical, and insilico approaches. Spectroscopic analysis revealed that EMB-modified protein undergoes conformational alterations. Furthermore, in vitro racemization studies of the MurI protein suggest that EMB decreases its functional activity. Docking studies revealed that EMB interacts with most of the active residues at the binding site and blocks the binding pocket. Overall, data suggests that EMB, a primary drug used for the treatment of tuberculosis (TB), acts as a competitive inhibitor of substrate for binding to mycobacterial MurI protein. The study also points out to our lacunae in understanding the site and mechanism of action of existing drugs. Furthermore, glutamate racemase is a conserved protein of the bacterial kingdom; therefore, ethambutol could be a promising candidate as a broad-spectrum antibiotic for many other bacterial diseases.

In-silico Hierarchical Approach for the Identification of Potential Universal Vaccine Candidates (PUVCs) from Neisseria gonorrhoeae.

OBJECTIVES: Resistance to the currently recommended extended-spectrum cephalosporins, which is used to treat Gonorrhea, is increasing continuously and leading to a threat of untreatable infection. It is, therefore, becoming extremely essential to search for new therapeutic strategies to control Gonorrhea. Vaccination may be considered as an effective control measure to control this disease, which is caused by Neisseria gonorrhoeae. METHODS: In-silico hierarchical approach was used to help identify candidate proteins of N. gonorrhoeae that might contribute significantly in vaccine research. In contrast to the conventional vaccine research which requires at least 10-12 years, the present approach would reduce the time period drastically and help to identify Potential Universal Vaccine Candidates (PUVCs). These proteins were further analyzed for the presence of T-cell and linear B-cell epitopes, by using HLAPred and ABCpred servers respectively, in order to facilitate the identification of Multi Epitope Peptide Vaccine Constructs. RESULTS: We have identified 23 non-host candidate proteins, using the proteomic information of four sequenced strains of N. gonorrhoeae namely FA 1090, TCDC_NG08107, NCCP11945 and MS11 and labeled them as PUVCs. Since all these identified 23 PUVCs contained both T cell and B cell epitopes, these have been further reiterated as PUVCs which could be used as promising leads for vaccine development. CONCLUSIONS: This hierarchical approach is the first comprehensive study to identify potential vaccine candidates which once utilized for vaccine development would surely serve as promising tools for effective control of Gonorrhea.

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress, inflammation, and cognitive impairment in rats: possible mechanisms of neuroprotection.

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic-clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABAA receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABAA receptor to produce anticonvulsant effects and to ameliorate cognitive impairment.

Naringin protects against kainic acid-induced status epilepticus in rats: evidence for an antioxidant, anti-inflammatory and neuroprotective intervention.

The effect of naringin, a bioflavanoid, with potent antioxidant activity was studied on kainic acid (KA)-induced seizures, cognitive deficit and oxidative stress. Rats were administered KA (10 mg/kg intraperitoneally (i.p.)) and observed for behavioral changes and incidence and latency of convulsions over 4 h. The rats were thereafter sacrificed and oxidative stress parameters like malondialdehyde (MDA) and glutathione (GSH) were estimated in the brain. The level of proinflammatory cytokine, tumor necrosis factor (TNF)-α was also determined in the rat brain. It was observed that pretreatment with naringin (20, 40, 80 mg/kg, i.p.) significantly (p<0.001) increased the latency of seizures as compared to the vehicle treated-KA group. Naringin (40, 80 mg/kg) also significantly prevented the increase in MDA and fall in GSH levels due to KA. In addition, naringin dose-dependently attenuated the KA-induced increase in the TNF-α levels of brain. The pretreatment with naringin also significantly increased retention latency in the passive avoidance task. This shows that naringin reduced the cognitive deficit induced by KA. The results of our study suggest that naringin has therapeutic potential since it suppresses KA-induced seizures, cognitive impairment and oxidative stress in the brain. These neuroprotective effects are a result of its antioxidant and anti-inflammatory activity.

An insight into the drug resistance profile & mechanism of drug resistance in Neisseria gonorrhoeae.

Among the aetiological agents of treatable sexually transmitted diseases (STDs), Neissseria gonorrhoeae is considered to be most important because of emerging antibiotic resistant strains that compromise the effectiveness of treatment of the disease - gonorrhoea. In most of the developing countries, treatment of gonorrhoea relies mainly on syndromic management rather than the aetiological based therapy. Gonococcal infections are usually treated with single-dose therapy with an agent found to cure > 95 per cent of cases. Unfortunately during the last few decades, N. gonorrhoeae has developed resistance not only to less expensive antimicrobials such as sulphonamides, penicillin and tetracyclines but also to fluoroquinolones. The resistance trend of N. gonorrhoeae towards these antimicrobials can be categorised into pre-quinolone, quinolone and post-quinolone era. Among the antimicrobials available so far, only the third-generation cephalosporins could be safely recommended as first-line therapy for gonorrhoea globally. However, resistance to oral third-generation cephalosporins has also started emerging in some countries. Therefore, it has become imperative to initiate sustained national and international efforts to reduce infection and misuse of antibiotics so as to prevent further emergence and spread of antimicrobial resistance. It is necessary not only to monitor drug resistance and optimise treatment regimens, but also to gain insight into how gonococcus develops drug resistance. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea.

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis.

BACKGROUND: Screening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world. METHODS: Prevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay. RESULTS: We detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission. CONCLUSIONS: The in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Screening of natural compounds that targets glutamate racemase of Mycobacterium tuberculosis reveals the anti-tubercular potential of flavonoids.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB), a highly infectious disease accounting for nearly 1.5 million deaths every year and has been a major global concern. Moreover, resistance to anti-TB drugs is an arduous obstacle to effective prevention, TB care and management. Therefore, incessant attempts are being made to identify novel drug targets and newer anti-tubercular drugs to fight with this deadly pathogen. Increasing resistance, adverse effects and costly treatment by conventional therapeutic agents have been inclining the researchers to search for an alternative source of medicine. In this regard natural compounds have been exploited extensively for their therapeutic interventions targeting cellular machinery of MTB. Glutamate racemase (MurI) is an enzyme involved in peptidoglycan (PG) biosynthesis and has become an attractive target due to its moonlighting property. We screened various classes of natural compounds using computational approach for their binding to MTB-MurI. Shortlisted best docked compounds were evaluated for their functional, structural and anti-mycobacterial activity. The results showed that two flavonoids (naringenin and quercetin) exhibited best binding affinity with MTB-MurI and inhibited the racemization activity with induced structural perturbation. In addition, fluorescence and electron microscopy were employed to confirm the membrane and cell wall damages in mycobacterial cells on exposure to flavonoids. Together, these observations could provide impetus for further research in better understanding of anti-tubercular mechanisms of flavonoids and establishing them as lead molecules for TB treatment.

Intrinsically disordered protein from a pathogenic mesophile Mycobacterium tuberculosis adopts structured conformation at high temperature.

Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.

Comparison of mammalian cell entry operons of mycobacteria: in silico analysis and expression profiling.

Mammalian cell entry (mce) operons, implicated in the entry of mycobacteria into host cells, are present in pathogenic and saprophytic species. It is likely that the genes in these operons have functions other than those required for entry into host cells. Using in silico analysis we have identified domains within the mce operons that might justify their occurrence in saprophytic species like Mycobacterium smegmatis. Our analysis identified in addition to the mce domain, the presence of the Ttg2B and Ttg2C domains, typical of proteins involved in transport. We have also analysed and compared the expression profile between mce operons of Mycobacterium tuberculosis, Mycobacterium bovis and M. smegmatis under different growth conditions. In case of M. smegmatis, each operon presented domain truncation for at least one gene. We observe differential expression among the operons in M. smegmatis growing under different culture conditions. Bacilli growing in nutritionally rich medium with aeration, only the mce4 operon was expressed while during stationary phase of a standing culture, all four mce operons were expressed. In M. bovis, in addition to the absence of the mce3 operon, several protein domains encoded by the other operons were truncated. We detected expression of the mce2 operon in the exponential and stationary growth phase, while the mce1 operon was only expressed in the stationary growth phase. Differential expression of mce operons and their redundancy in the genome of the majority members of mycobacteria are discussed in view of our results.

Cefepime versus ceftazidime as empirical therapy for fever in neutropenic patients with haematological malignancies.

An open randomized comparative study was conducted to evaluate the efficacy of Cefepime (2 gm iv. 8 hr.) vs. ceftazidime (2 gm iv. every 8 hr.) in empirical therapy of febrile neutropenic patients. A total of 40 eligible febrile episodes were randomized to be treated with study regimen. Twenty febrile episodes were treated with cefepime and 20 were treated with ceftazidime. The two groups were comparable in terms of age, sex, height, underlying neoplasm, number of pretherapy neutrophils, duration of neutropenia. The overall therapeutic success rate of cetepime group (60%) was comparable to that of ceftazidime group (55%). The results of this study suggest that cefepime is an effective and safe agent in empirical therapy of febrile episode in neutropenic patient and its efficacy is comparable with that of ceftazidime.