Drug-Resistance of Pseudomonas aeruginosa
The treatment of P. aeruginosa infections is extremely problematic because of the intrinsic resistant to a broad range of antibiotics.6 It can easily develop resistance by means of several mechanisms which are associated with low membrane permeability, efflux pump and genetic mutations.10 Multi-factorial mechanism of antibiotic resistance in P. aeruginosa is based on low outer membrane permeability for the antibiotics, multiple antibiotic modifying enzymes like ß-lactamases, metallo-ß-lactamases and aminoglycoside modifying enzyme, efflux pumps. Such as, MexAB-OprM, MexEF-OprN, MexCD-OprJ and MexXY-OprM and acquisition of encoded antibiotic resistance genes via plasmid and chromosomal mutations.6 The first resistance mechanism is low outer membrane permeability, which serves to decrease the rate of uptake of most antibiotics and the second mechanisms is energy dependent multidrug efflux and ß-lactamase.
The resistance rates of P. aeruginosa strains are significantly higher than the other gram-negative pathogens because it has large and versatility genome which are able to develop new resistance mechanisms to antibiotics and also contributes its pathogenicity. They also cause higher rate of mortality compare to other gram negative bacteria.
The therapy for P. aeruginosa infections is more challenging since it has also multi-drug resistance which means the bacteria has resistance more than two of the antibiotics that were once effective for the combating of infectious. Multi-drug resistance is making combination therapy of P. aeruginosa infections useless, difficult and unsuccessful.12 Unfortunately, extensive use of antibiotics are leading to increase the amount of drug-resistant strains of P. aeruginosa.
Hopefully, there are several alternative approaches in order to overcome difficulties in treating P. aeruginosa infections.
The Type III Secretion System and Exotoxin S
There are numerous virulence factors that contribute to the pathogenicity of P. aeruginosa. A major virulence factor of P. aeruginosa is the type III secretion system (T3SS) that is responsible of secretion effector toxins.13 These toxins are ExotoxinS (ExoS), ExoT, ExoU and ExoY which play an important roles in the pathogenesis of the bacteria.13b T3SS is a needle-like complex structure and has five groups of proteins, which are the needle complex, the translocation apparatus, regulator proteins, chaperons and effector toxins.
Only four effector proteins have been identified in T3SS of P. aeruginosa. Most strains of P. aeruginosa do not have the four effector encoding genes.14 For this reason, strains of P. aeruginosa have either ExoS or the ExoU gene but not both of them. This characteristic feature can help to define phenotype of strains during the infection.14 ExoS is a bifunctional toxin and has GTPase activating protein (GAP) activity and adenosine diphosphate ribosyl transferase (ADPRT) activity.15 The amino-terminus ExoS possess membrane localization domain (MLD) that is responsible for the temporary localization of ExoS to the plasma membrane of the host cell.14 This localization of ExoS is very important for the useful modification of its substrates. Targets of GTP domain of ExoS are Rho, Rac and Cdc42.14 GTP domain of ExoS causes disruption of the actin cytoskeleton of the host cell. ADPRT domain of ExoS has a wide number of negative effects on the host cell such as cell death, disruption of the actin cytoskeleton via cell rounding, inhibition of DNA synthesis and endocytosis. Both domain of ExoS lead to irreversible damage to the host cell by disruption of the cytoskeleton.
STO1101 and ME0569: Inhibitors of ExoS-ADPRT
A recent study published by our group in collaboration with researchers at Karolinska Institute has identified STO1101 and ME0569 (Figure 1) as potent inhibitors of ExoS-ADPRT.
STO1101 is a competitive inhibitor of ExoS with an IC50 value of 19 µM. In the structure of this molecule, there are two adjacent rings which are pyrimidone and cyclopente[b]thiophene and this ring system was substituted with propionic acid.
The IC50 value of ME0569 is 25 µM and it has quinazolinone ring which substituted with butyric acid.
Popular scientific summary including social and ethical aspects
Pseudomonas aeruginosa is one of the most common pathogen that causes of serious infections such as pneumonia, meningitis, soft tissue infections, chronic lung infections and corneal infections. P. aeruginosa infections associated with high rate of morbidity and mortality especially among immunocompromised individuals such as cystic fibrosis (CF), burn wound, or cancer.11 Due to intrinsic resistance to a broad range of antibiotics, P. aeruginosa infections are very difficult to eradicate compared with other gram-negative pathogens infections.6 Currently, P. aeruginosa infections may be treated by a combination of anti-pseudomonal agents in order to tackle resistance issue. However, this standard combination therapy remains problematic and not effective, as it leads to the increase of the drug-resistant strains.18 Therefore, there are not many useful anti-pseudomonal drugs, P. aeruginosa infections are still one of the most dangerous infection disease in clinic. To combat the difficulties in treating P. aeruginosa infections, there are several approaches to develop new anti-pseudomona drugs. Targeting virulence factors, the ability of the bacteria to causes disease, is one of those novel strategies to fight against antibiotic resistance by disarming the bacteria.11 Virulence factors of P. aeruginosa are responsible for the severity of infections because they cause irreversible host cell damage.11 Especially exotoxins and proteases are associated with cell and tissue damage by disrupting the cytoskeletal structure and to develop of chronic infections.11 P. aeruginosa has five protein secretion systems and among them, the type III secretion system transfers toxins (ExoS-T-U-Y) into the host cell.15 ExoS is a bifunctional enzyme and possesses a GTPase-activating protein (GAP) activity and a ADP-ribosyl transferase (ADPRT) activity.15 These activities work to disrupt the actin cytoskeleton and cause to cell death. The ADPRT domain of ExoS is responsible for the irreversible cell damage and it is highly toxic to cultured cells.
Social and ethical aspects
Infection diseases are still major problem of clinic and P. aeruginosa causes severe and life-threating infections. Infections of P. aeruginosa are clinically challenged since it can readily develop antibiotic resistance during the therapy.18 If the resistance occurs during the therapy, it will cause the length of hospital stay, additional medical procedures, surgery, chronic care and overall cost of antibiotics and even it will lead to the death of patients.18 The high rate of drug-resistant strains of P. aeruginosa is increasing health threats facing the nation.
For this reason, there is an urgent need to develop new therapeutic agents against this pathogen for the combating P. aeruginosa infections. This project will help us to develop a new effective chemical probe which will be used as a chemical tool for the discovery of anti-pseudomonal drugs. Because of that this project is very important for the public health.
General. All reactions were carried out under nitrogen atmosphere. Chemicals and reagents were purchased from Aldrich, Alfa Aesar, AK Scientific, Matrix Scientific, or Apollo Scientific. Organic solvents were dried using the dry solvent system (Glass Contour Solvent Systems, SG Water USA) except CH3CN, EtOH and PhCH3, which were dried over activated molecular sieves 3 Å or 4 Å. Flash chromatography was performed on Biotage Isolera One using appropriate SNAP Cartridge KP-Sil or SNAP Ultra HP-Sphere 25µm Cartridge and UV absorbance at 254 nm. TLC was performed on Silica gel 60 F254 (Merck) with detection by UV light unless staining solution is mentioned. Preparative HPLC separation were performed on Gilson System HPLC, using a VP 250/21 NUCLEODUR C18 column HTEC 5 μm with a flow rate 18 mL/min, detection at 214 nm and eluent system: A. aq. 0.075% HCOOH, and B. 0.075% HCOOH in CH3CN. The NMR spectra were recorded at 298 K on Bruker-DRX 400 MHz and 600 MHz using the residual peak of the solvent DMSO-d6 (δH 2.50 ppm) or CDCl3 (δH 7.26 ppm) as internal standard for 1H, and DMSO-d6 (δc 39.50 ppm) and CDCl3 (δc 77.16 ppm) as internal standard for 13C. LCMS were recorded by detecting positive/negative ion (EC+/EC-) with an electrospray Water Micromass ZG 2000 instrument using XTerra MS C18 (5 μm 19×50 mm column) and H2O/CH3CN (0.2% HCOOH) as the eluent system, or with Agilent 1290 infinity – 6150 Quadrupole using YMC Triart C18 (1.9 μm 20×50 mm column) and H2O/CH3CN (0.1% HCOOH) as the eluent system.
Table of contents :
List of Abbreviations
1.1. Pseudomonas aeruginosa
1.2 Drug-Resistance of Pseudomonas aeruginosa
1.3 The Type III Secretion System and Exotoxin S
1.4 STO1101 and ME0569: Inhibitors of ExoS-ADPRT
1.5 Aim of the diploma work
2. Popular scientific summary including social and ethical aspects
2.1 Popular scientific summary
2.2 Social and ethical aspects
3.1 Chemistry Section
3.2 Biology Section
3.2.1 Dose-response experiment:
4. Results and Discussion
6. Future Plan
7. Appendix I
8. Appendix II
9. Appendix III