Target For Antimicrobial Chemotherapy Research Articles

Antimicrobial resistance: a Biochemical Society position statement

Antimicrobial resistance: a Biochemical Society position statement

Functionalized Chitosan Nanomaterials: A Jammer for Quorum Sensing.

The biggest challenge in the present-day healthcare scenario is the rapid emergence and spread of antimicrobial resistance due to the rampant use of antibiotics in daily therapeutics. Such drug resistance is associated with the enhancement of microbial virulence and the acquisition of the ability to evade the host’s immune response under the shelter of a biofilm. Quorum sensing (QS) is the mechanism by which the microbial colonies in a biofilm modulate and intercept communication without direct interaction. Hence, the eradication of biofilms through hindering this communication will lead to the successful management of drug resistance and may be a novel target for antimicrobial chemotherapy. Chitosan shows microbicidal activities by acting electrostatically with its positively charged amino groups, which interact with anionic moieties on microbial species, causing enhanced membrane permeability and eventual cell death. Therefore, nanoparticles (NPs) prepared with chitosan possess a positive surface charge and mucoadhesive properties that can adhere to microbial mucus membranes and release their drug load in a constant release manner. As the success in therapeutics depends on the targeted delivery of drugs, chitosan nanomaterial, which displays low toxicity, can be safely used for eradicating a biofilm through attenuating the quorum sensing (QS). Since the anti-biofilm potential of chitosan and its nano-derivatives are reported for various microorganisms, these can be used as attractive tools for combating chronic infections and for the preparation of functionalized nanomaterials for different medical devices, such as orthodontic appliances. This mini-review focuses on the mechanism of the downregulation of quorum sensing using functionalized chitosan nanomaterials and the future prospects of its applications.

Roles of Regulatory RNAs for Antibiotic Resistance in Bacteria and Their Potential Value as Novel Drug Targets.

The emergence of antibiotic resistance mechanisms among bacterial pathogens increases the demand for novel treatment strategies. Lately, the contribution of non-coding RNAs to antibiotic resistance and their potential value as drug targets became evident. RNA attenuator elements in mRNA leader regions couple expression of resistance genes to the presence of the cognate antibiotic. Trans-encoded small RNAs (sRNAs) modulate antibiotic tolerance by base-pairing with mRNAs encoding functions important for resistance such as metabolic enzymes, drug efflux pumps, or transport proteins. Bacteria respond with extensive changes of their sRNA repertoire to antibiotics. Each antibiotic generates a unique sRNA profile possibly causing downstream effects that may help to overcome the antibiotic challenge. In consequence, regulatory RNAs including sRNAs and their protein interaction partners such as Hfq may prove useful as targets for antimicrobial chemotherapy. Indeed, several compounds have been developed that kill bacteria by mimicking ligands for riboswitches controlling essential genes, demonstrating that regulatory RNA elements are druggable targets. Drugs acting on sRNAs are considered for combined therapies to treat infections. In this review, we address how regulatory RNAs respond to and establish resistance to antibiotics in bacteria. Approaches to target RNAs involved in intrinsic antibiotic resistance or virulence for chemotherapy will be discussed.

Chitosan nanoparticles enhances the anti-quorum sensing activity of kaempferol

Quorum sensing (QS) is a cell density dependent expression of species in bacteria mediated by compounds called autoinducers (AI). Several processes responsible for successful establishment of bacterial infection are mediated by QS. Inhibition of QS is therefore being considered as a new target for antimicrobial chemotherapy. Flavonoid compounds are strong antioxidant and antimicrobial agents but their applications are limited due to their poor dissolution and bioavailability. Our objective was to investigate the effect of kaempferol loaded chitosan nanoparticles on modulating QS mediated by AI in model bioassay test systems. For this purpose, kaempferol loaded nanoparticles were synthesized and characterized in terms of hydrodynamic diameter, hydrogen bonding, amorphous transformation and antioxidant activity. QS inhibition in time dependent manner of nanoparticles was measured in violacein pigment producing using the biosensor strain Chromobacterium violaceum CV026 mediated by AI known as acylated homoserine lactone (AHL). Our results indicated that the average kaempferol loaded chitosan/TPP nanoparticle size and zeta potential were 192.27±13.6nm and +35mV, respectively. The loading and encapsulation efficiency of kaempferol into chitosan/TPP nanoparticles presented higher values between 78 and 93%. Kaempferol loaded chitosan/TPP nanoparticle during the 30 storage days significantly inhibited the production of violacein pigment in Chromobacterium violaceum CV026. The observation that kaempferol encapsulated chitosan nanoparticles can inhibit QS related processes opens up an exciting new strategy for antimicrobial chemotherapy as stable QS-based anti-biofilm agents.

Reduction of the Peptidoglycan Crosslinking Causes a Decrease in Stiffness of the Staphylococcus aureus Cell Envelope

We have used atomic-force microscopy (AFM) to probe the effect of peptidoglycan crosslinking reduction on the elasticity of the Staphylococcus aureus cell wall, which is of particular interest as a target for antimicrobial chemotherapy. Penicillin-binding protein 4 (PBP4) is a nonessential transpeptidase, required for the high levels of peptidoglycan crosslinking characteristic of S. aureus. Importantly, this protein is essential for β-lactam resistance in community-acquired, methicillin-resistant S. aureus (MRSA) strains but not in hospital-acquired MRSA strains. Using AFM in a new mode for recording force/distance curves, we observed that the absence of PBP4, and the concomitant reduction of the peptidoglycan crosslinking, resulted in a reduction in stiffness of the S. aureus cell wall. Importantly, the reduction in cell wall stiffness in the absence of PBP4 was observed both in community-acquired and hospital-acquired MRSA strains, indicating that high levels of peptidoglycan crosslinking modulate the overall structure and mechanical properties of the S. aureus cell envelope in both types of clinically relevant strains. Additionally, we were able to show that the applied method enables the separation of cell wall properties and turgor pressure.

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

Menthol diminishes Staphylococcus aureus virulence-associated extracellular proteins expression

Staphylococcus aureus is a significant human pathogen that is the major cause of a broad spectrum of illnesses, ranging from minor skin infections to life-threatening deep tissue infections and toxinosis. The ability of the organism to cause such a broad range of infections is, to a great extent, attributed to the secretion of a myriad of virulence-related extracellular proteins. Therefore, virulence as a target for antimicrobial chemotherapy has gained great interest. Menthol is a monocyclic terpene alcohol that occurs naturally in plants of the Mentha species lacking anti-S. aureus activity. In this paper, we demonstrate via hemolytic activity assays, tumor necrosis factor release assays, Western blot assays, and real-time reverse transcription-PCR assays that low concentrations of menthol can markedly inhibit the expression of α-hemolysin, enterotoxins A and B, and toxic shock syndrome toxin 1 in S. aureus. Our results indicate that menthol may be useful in managing S. aureus infections when used in combination with β-lactam antibiotics, which can often increase S. aureus toxin secretion when used at subinhibitory concentrations. In addition, the menthol basic structure has potential applications in the development of new anti-virulence drugs.

Dietary phytochemicals as quorum sensing inhibitors

Quorum sensing (QS) is a cell density dependent expression of species in bacteria mediated by hormone like compounds called autoinducers (AI). Several processes responsible for successful establishment of bacterial infection are mediated by QS. Inhibition of QS is therefore being considered as a new target for antimicrobial chemotherapy. Dietary phytochemicals are secondary metabolites in plants known to have several health benefits including antimicrobial activity. However, their ability to inhibit QS has never been studied. Our objective was to investigate the effect of sub-lethal concentrations (SLC) of bioactive dietary phytochemical extracts from common dietary fruit, herb and spice extracts on modulating QS mediated by AI in model bioassay test systems. QS inhibition was measured in violacein pigment producing Chromobacterium violaceum O26 (CVO26) and CV 31532 system, mediated by AI known as acylated homeserine lactone (AHL). We also investigated the effect of the sub-lethal concentrations of the extracts on swarming motility of pathogens Escherichia coli (EC)O157 :H7 and Pseudomonas aeruginosa (PA-01). Our results indicate that all extracts significantly inhibited quorum sensing. The mechanism of inhibition appeared to be combination of interfering with AHL activity and modulating the synthesis of AHL's. Our results also indicated that various phytochemical extracts which inhibited QS also inhibited swarming of pathogenic bacteria, known to be modulated by QS. The observation that phytochemicals from foods can inhibit QS related processes opens up an exciting new strategy for antimicrobial chemotherapy and lead to the discovery of new category of antibiotics which can overcome the issues related to antimicrobial resistance.

The yjeQ Gene Is Required for Virulence of Staphylococcus aureus

Gene products required for in vivo growth and survival of Staphylococcus aureus and other pathogens represent new targets for antimicrobial chemotherapy. In this study we created a Staphylococcus aureus yjeQ deletion strain and tested its virulence using a mouse kidney abscess infection model. The yjeQ deletion strain was compromised for growth in vitro and severely attenuated for virulence. We concluded that yjeQ is an attractive and novel new drug target.

Large-Scale Identification of Genes Required for Full Virulence ofStaphylococcus aureus

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Effect of srtA and srtB gene expression on the virulence of Staphylococcus aureus in animal models of infection.

The role that the surface proteins anchored by the srtA and srtB gene products play in the ability of Staphylococcus aureus bacteria to establish infection was investigated in several animal models. Wild-type and corresponding mutants with deletions of the srtA and/or srtB genes were used in murine acute lethal infection, septic arthritis, kidney infection and rat endocarditis models. The LD(50) of the wild-type and srtB- knockout were comparable and approximately two- to four-fold lower than the required inoculum of the srtA- and srtA-B- strains. This difference was exhibited as a two-fold greater mortality at the highest inoculum. The wild-type strain established arthritic inflammation in over 90% of the animals with a maximum arthritic index of 6.5 by days 17-21. The srtB- knockout was able to cause inflammation in 70-80% of the mice, but with a lower index of 3.0. Both the srtA- and srtA-B- strains appeared to be less virulent in this model with arthritic indices of around 0.5 and only 20% of the animals with inflammation. Strains with the srtA mutation achieved statistically significant lower titres than wild-type in kidneys of mice after intravenous infection. Mean bacterial counts in cardiac vegetations were significantly higher for the wild-type and srtB- strain compared with the srtA- and srtA-B- strains. Results from this study substantiate the role of the srtA gene product in the establishment of infections and further studies are warranted to define and exploit this as a target for antimicrobial chemotherapy.

Peptide Deformylase Inhibitors, Potential for a New Class of Broad Spectrum Antibacterials

Peptide deformylase (PDF) has emerged as one of the most promising new targets for antimicrobial chemotherapy. PDF is an essential bacterial metalloenzyme that deformylates the N-formylmethionine of newly synthesised bacterial polypeptides. Although the enzyme has been known for over 30 years, it has proved difficult to isolate and characterise due to its apparent instability. However, recent progress in understanding the structure and function of PDF has greatly facilitated the drug discovery process. In this article we review the potential of PDF as an antibacterial target and highlight progress in the development of inhibitors of PDF. The field has evolved particularly rapidly through the use of high throughput screening and combinatorial techniques and the diverse series of structures exploited previously in other metalloenzyme programmes. Although many different structural classes of compounds have been reported as inhibitors of PDF, the real challenge has been in obtaining molecules with good a ntibacterial activity. Several PDF inhibitors have now been described that demonstrate potent in vitro antibacterial activity against a range of Gram-negative and Gram-positive drug resistant pathogens. Results in stringent animal models of infection suggest that for some of these molecules good in vitro activity translates into good in vivo efficacy. One of the most encouraging compounds reported, BB-83698, has shown activity against Streptococcus pneumoniae in both the neutropenic mouse thigh and lung infection models. These data suggest that PDF inhibitors are a promising new class of antibacterial agents with opportunities to treat respiratory and other bacterial infections. Keywords: peptide deformylase, broad spectrum antibacterial, bacterial polypeptode, bb-83698, pdf inhibitor

Role of the oxyanion binding site and subsites S1 and S2 in the catalysis of oligopeptidase B, a novel target for antimicrobial chemotherapy.

Oligopeptidase B is a member of a novel serine peptidase family, found in Gram-negative bacteria and trypanosomes. The enzyme is involved in host cell invasion, and thus, it is an important target for drug design. Oligopeptidase B is specific for substrates with a pair of basic residues at positions P1 and P2. The sensitivity of substrates to high ionic strength suggests that the arginines interact with the carboxylate ions of the enzyme. On the basis of a three-dimensional model, two carboxyl dyads (Asp460 and Asp462 and Glu576 and Glu578) can be assigned as binding sites for arginines P1 and P2, respectively. The dyads are involved in several events: (i) substrate binding, (ii) substrate inhibition at high substrate concentrations (different inhibitory mechanisms were demonstrated with substrates bearing one and two arginine residues), (iii) enzyme activation at millimolar CaCl2 concentrations with substrates having one arginine, and (iv) interaction of Ca2+ with the dyads which simplified the complex pH dependence curves. Titration with a product-like inhibitor revealed the pK(a) of the carboxyl group that perturbed the pH-kcat/Km profiles. The OH group of Tyr452 is part of the oxyanion binding site, which stabilizes the transition state of the reaction. Its role studied with the Tyr452Phe variant indicates that (i) the catalytic contribution of the OH group depends on the substrate and (ii) the catalysis is, unusually, an entropy-driven process at physiological temperature. The NH group of the scissile peptide bond accounts for the deviation of the reaction from the Eyring plot above 25 degrees C, and for abolishing potential nonproductive binding.