Research Ideas and Outcomes :
Research Idea
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Corresponding author: Raphael Lartey Abban (raphaelabban@yahoo.com), Isawumi Abiola (isawumiabiola@gmail.com)
Academic editor: Editorial Secretary
Received: 23 May 2023 | Accepted: 13 Oct 2023 | Published: 19 Oct 2023
© 2023 Raphael Abban, Sarpong Kwabena, Samuel Duodu, Lydia Mosi, Isawumi Abiola
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Abban RL, Kwabena S, Duodu S, Mosi L, Abiola I (2023) Targeting Proteus mirabilis BAM Complex Proteins for Development of Novel Antibiotics. Research Ideas and Outcomes 9: e106849. https://doi.org/10.3897/rio.9.e106849
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Urinary tract infections (UTIs) are frequent hospital-acquired infection, with Escherichia coli and Proteus mirabilis accounting for 90% of complicated UTIs. Emergence of multidrug-resistant (MDR) bacteria have complicated the treatment UTIs. P. mirabilis related UTIs has been associated with the production of urinary stones and long-term infections in patients with catheters. P. mirabilis and other uropathogens constitute a largely unexplored pathogen group. The pathogen is resistant to most antibiotics as a result of its impermeable outer membrane (OM). The β-barrel assemble machinery folds and inserts outer membrane proteins; however, there are no antibiotics targeting the OM assemble in clinical use currently. Therefore, this study seeks to identify drugs that will inhibit the activity of P. mirabilis B complex proteins and also determine their effects on P. mirabilis OM biogenesis. This would be achieved by screening approved drugs against the P. mirabilis Bam complex using computer-based in silico screening and cellular-based assays. First, the binding effects of drugs on P. mirabilis B complex proteins will be determined using docking algorithms. The antimicrobial and antivirulence activity of selected drugs from in silico analysis will be screened against MDR P. mirabilis. Finally, the effect of active drug(s) on the OM biogenesis of wild-type P. mirabilis and mutant P. mirabilis will be determined using peptide nucleic acids (PNA). Western lot analysis will be used to determine the abundance of proteins involved in OM biogenesis. Successful completion of this study will lead to the identification of novel antibiotics against MDR P. mirabilis and associated mechanisms while providing the foundation for future research endeavours on other uropathogens.
rinary tract infections, Proteus mirabilis, antibiotics, urinary stones, outer membrane, β-barrel assemble machinery, Peptide Nucleic Acid
Proteus spp. are Gram-negative rod-shaped bacteria belonging to the Enterobacterales order and the Morganellaceae family. They are found naturally in the guts of animals and humans, and their existence in the surroundings is attributed to faecal contamination (
Global public health is seriously threatened by antimicrobial resistance (AMR). The spread of AMR is aided by the misuse of antibiotics (
The OM is made up of β-barrel proteins that facilitate the binding and folding of the β-barrel complex (BAM complex) (
On the surface of Gram-negative bacteria are adhesion proteins which aid in the interaction between the pathogen and host by initiating infections and coloniing host cells
Drug repurposing and repositioning are effective in the development of new and effective drugs to circumvent the challenges of AMR. This approach saves time and its cost-effective (
The research objectives include screening for drugs with activity against P. mirabilis B complex proteins using in silico computer-based method; determination of antimicrobial and antivirulence activities of selected drugs on P. mirabilis using in vitro drug assays; and the effects of identified drugs on the OM biogenesis of wild-type and mutant P. mirabilis would be determined. These objectives will be achieved as described (Fig.
Urinary Tract Infections (UTI)
UTI is a serious health problem caused by bacteria. Most hospitals regularly observe this globally, with 150 million cases annually (
Uropathogens and Antimicrobial Resistance
Bacteria and fungi are common causative pathogens of UTIs, which might be uncomplicated complicated. Complicated UTIs are caused by E. coli, Klebsiella pneumonia, Staphylococcus saprophyticus, group B streptococcus (GBS), Pseudomonas aeruginosa, Enterococcus faecalis, P. mirabilis, S. aureus and Candida species. Uncomplicated UTIs are caused by Uropathogenic E. coli, Enterococci, K. pneumonia, GBS, P. aeruginosa, Candida species and S. aureus (
Drug Repurposing (DR)
DR involves identifying new uses for experimental, approved, abandoned and discontinued drugs to treat diseases. It is an innovative strategy that replaces traditional drug discovery, as it reduces time, money, safety issues and the likelihood of failure associated with traditional drug discovery. DR can be done experimentally or by in silico approach. The experimental approach involves screening drugs for therapeutic effects using biological assays. It requires in vitro or/and in vivo screening of drugs against the cellular components or drug targets. In the in silico approach, available chemical libraries from databases are virtually screened against protein targets using bioinformatics tools. Bioactive molecules are identified based on the molecular interaction of drugs with these targets (
Computational versus Experimental DR
Compared to experimental approaches, in silico DR reduces time and risk of failure and is also cost-effective. The drawback in the experimental approach is that it requires accurate details about drug targets, drugs and disease phenotypes (
B Complex Protein
B complex is the central machinery that assembles the unfolded OM proteins to create a highly impermeable barrier via the sur A chaperone (
P. miralbilis is a critical pathogen threatening human health, especially for UTI patients. The last-resort treatments for MDR-bacteria, third-generation cephalosporins and carbapenems, have not been effective (
This study will enhance better understanding of the function of B Complex proteins in P. mirabilis. The ex-vivo assay of field isolates will provide a broader perspective of the bacterium's in vivo behaviour. , the study will provide additional insights into alternative therapeutic approach to design future antibiotics (such as antibacterial, antivirulence and antisense drugs). Finally, the prioritiation of the B complex proteins and elucidation of drugs' mechanism of action will add more value to the knowledge of drug development.
Research Question 1: Are there drugs which inhibit the activity of Proteus mirabilis B complex proteins?
OBJECTIVE 1: Screen for drugs with activity against P. mirabilis B complex proteins.
Homology Modelling Structures of P. mirabilis B ABCDE
B Complex protein sequence templates will be obtained from National Centre for Biotechnology Information (NCBI). Homology models will be generated based on the pairwise alignment of template and target sequences. Homology models of Bam A-E will be built in Modeller 10.2 utiliing templates with significant levels of sequence identity and coverage. DOPE scores will be used to rank models. ProSA-web, PROCHECK and ERRAT will be used to evaluate the quality of the modelled proteins.
Ligands Preparation and Molecular Docking
The 3D structures of about 3000 inhibitors (1500 on-target and 1500 off-target inhibitors) with their respective IDs will be retrieved from the PubChem database https://pubchem.ncbi.nlm.nih.gov/ and African Natural Product Database (ANPD - http://african-compounds.org/anpdb/. PyRx version 0.8 (AutoDock Vina - https://pyrx.sourceforge.io/downloads will be used for docking experiments. The structure of the drug will be converted to pdbqt format and energy minimied using MMFF94 forcefield and Conjugate Gradient. Blind docking will be used, since the binding pockets of targets are unknown. PyMol will be used to display the docked structures. -8.0 kcal/mol will be binding energy cut-off point. The docked complexes will be loaded into LigPLOT+v1.45 to produce the ligand 2D interaction.
Pharmacokinetics Profiling
The SwissADME web resource (http://www.swissadme.ch/) will be used to determine the drug-likeness and pharmacokinetics properties of hit compounds as described by
Molecular Dynamics (MD) Simulation
To assess the stability of the system, MD simulation will be performed on the binding modes of the docked complexes as described by
OBJECTIVE 2: determine the antimicrobial and antivirulence activity of selected drugs against Proteus mirabilis.
Strains of Microorganisms: Proteus mirabilis resistant catheters isolates will be obtained from patients (men and women - 60 years old) in the intensive care unit (ICU). These samples were characteried by typing UreR gene with PCR and conventional biochemical assays by the AbiMosi Bacteria Culture of the Department of Biochemistry, Cell and Molecular Biology. The strains are resistant to more than one antibiotic (ciprofloxacin, ceftazidime, colistin and imipenem) used in treating UTIs. The resistant strain P. mirabilis (ATCC BAA-3089) will be used as a control strain. For all drug assays, gentamicin and meropenem would be used as control antibiotics, as they are antibiotics of choice for treating UTIassociated infections in the study area.
Antimicrobial Activity of Selected Drugs on Proteus mirabilis
Overnight cultures of P. mirabilis strains (108 CFU/ml expressed as (Number of colonies x Dilution Factor)/Volume of culture plated) will be inoculated into 10 ml of Mueller Hinton (MH) broth at 37⁰C and adjusted to Optical Density (OD) 600 nm of 0.5 MacFarland standard. About 50 µl of the culture will be transferred into each well of a 96-well plate containing LB broth (100 µl). Ten elected drugs from objective 1 (Artenimol, Dasatinib) at different concentrations (200 µg/ml-1000 µg/ml) will be introduced into the wells, incubated (24 h, 37⁰C, 180 rpm) and OD measured with microplate reader (OD 600 nm) and minimum inhibition concentration (MIC) determined as described by
Effect of Drugs on Proteus mirabilis Using Fluorescent Microscopy
The effect of the varying concentrations of drugs on P. mirabilis after incubation for 4 and 8 h will be determined via live/dead staining technique according to
Antivirulence Activity of Selected Drugs on P. mirabilis
HeLa cells will be seeded in 96-well plate with/without selected drugs (at different concentrations), infected with P. mirabilis and incubated (5% CO2, 37°C, 2 h). Non-adherent bacterial cells will be removed from the wells by washing three times with PBS. Cell cultures will be treated with 0.025% Triton X-100 (5 minutes, 37°C, 5% CO2) to separate and lyse the cell monolayer. The cell lysates will be diluted ten-fold and inoculated onto Trypticase Soy Agar (TSA). After incubation (24 h, 37°C), the colony forming unit (CFU/ml) will be calculated as described by
Effect of Drugs on Expression of adhesion genes (mrpA and pmfA)
10-100 cells/ml overnight cultures of P. mirabilis will be treated with ten selected drugs at 37⁰C and incubated at different time points (2, 8, 16, 24 and 48 h). Total RNA will be extracted from bacteria cells using RNAeasy extraction kit and cDNA synthesis synthesied with reverse-transcriptase. qRT-PCR will be conducted with 20 µl reaction consisting 2× Master Mix (SYBR Green Ampliqon), RNAase-free double distilled water, Primers (10 pM) and cDNA (5 ng/µl). The cycling condition will be: denaturation (95⁰C, 10 minutes), denaturation (95⁰C, 15 seconds, 44 cycles), annealing and elongation (54⁰C, 60 seconds). The internal control will be 16S rRNA and the primers designed using the Primer 3 tool. Triplicate samples will be analyed and the 2-ΔΔCT method will be used to determine the fold change in expression of mrpA and pmfA genes, as described by
Research Question 2: How does the drug interact and inhibit P. mirabilis B complex?
OBJECTIVE 3: determine the effect of active drugs on wild-type and mutant P. mirabilis outer membrane biogenesis. This objective determines changes in the OM pathway in wild-type and mutant P. mirabilis as a result of drug treatment. The expression of B complex proteins will be down modulated using Peptide Nucleic Acid (PNA) to validate the mode of action of drugs. Gentamicin and meropenem would be used as control antibiotics, as they are antibiotics of choice for treating UTIs associated infections in the study area.
Antisense PNA Design
Specific antisense PNA for B ABCDE genes in P. mirabilis will be synthesied by semi-automated parallel synthesis using the ABIMED robot described by
Generation of mutant P. mirabilis
P. mirabilis culture in LB broth with previouslystated antibiotics and different concentrations (2 µM, 4 µM, 6 µM, 8 µM, 10 µM) of gene-specific PNA will be co-culture and incubated at 37⁰C overnight. For positive control, P. mirabilis will be cultured with an antisense PNA as a positive control while a non-specific antisense PNA as negative control. Bacterial cells will be lysed and analyed by estern lotting to confirm the specificity of antisense PNA as described by
MIC of Active Drugs against wild-type and mutant P. mirabilis
Overnight cultures of Wild-type and mutant P. mirabilis strains (108 CFU/ml expressed as Number of colonies x Dilution Factor)/Volume of culture plated) will be inoculated into LB broth (10 ml) at 37⁰C and adjusted to Optical Density (OD) 600 of 0.5 MacFarland standard. About 50 µl of the culture will be pipetted into each well of a 96-well plate containing LB broth (100 µl). Serial dilutions of four active drugs will be prepared in the broth in a final assay volume of about 100 µl. About 50 µl of bacterial culture will be added to the seriallydiluted drugs and incubated (37⁰C, 24 h). The bacterial growth will be monitored using microplate reader and MIC determined as previously indicated.
Sample Preparation for Western lot Analysis
Using the same sample from above, 25 µl of the samples will be reconstituted with 1U benzonase at an OD of 600 and incubated (27⁰C, 10 min). The samples will be mixed with 25 µl β-mercaptoethanol supplemented with Laemilli sample buffer. The samples will be boiled for 10 min to measure total OMP levels in estern blot analysis (
Abundance of OM Protein in both wild-type and mutant P. mirabilis
Samples from bacteria strains will be loaded on a sodium dodecyl sulphate polyacrylamide gel electrophoresis at 120 V as described by
DATA ANALYSIS
All experiments will be repeated thrice with average and standard deviations calculated. One-way analysis of variance (ANOVA) will be used for comparative mean analysis as indicated by Tukeys Post hoc analysis in Graph Pad Prism. The docked complex of Bam A with a ligand is shown in Fig.
The authors appreciate AMR Research Group led by Dr Isawumi Abiola at the Department of Biochemistry, Cell and Molecular Biology and West African Centre for Cell Biology of Infectious Pathogens at the University of Ghana for training.
West African Centre for Cell Biology of Infectious Pathogens, Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon, Accra, Ghana