Molecular Bioscience , Protein Engineering, Protein Modelling, Recombinant Protein Expression, Bacterial AB Binary Toxins.
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Scopus Publications
Scopus Publications
Structural modelling and in silico analysis of a thiol-activated cytolysin produced by Bacillus thuringiensis Husam Sabah Auhim, Nisreen Hadi Odaa, Ismail Taban Iraqi Journal of Science, 2026 Bacillus thuringiensis (Bt) is a soil-dwelling, Gram-positive bacterium that produces proteins with specific toxic effects against invertebrate organisms. These toxins specifically target insects. This study examined a hypothetical Bt protein for its potential toxicity to mammals. The hypothetical protein was analyzed using various bioinformatics tools, including NCBI BLAST and conserved domain tools. The physicochemical properties were assessed using ExPasy ProtParam, and homology models were generated through automated services. The analysis revealed that the hypothetical protein is a thiol-activated cytolysin, a pore-forming toxin that targets mammalian cells with a signature motif. The toxin has a molecular weight of 52.6 kDa, and its instability index is 25.73, indicating it is stable under physiological conditions. Furthermore, the toxin is expected to exhibit thermal stability, as evidenced by its aliphatic index of 79.52. Three homology modeling services were used, homology modeling identified anthrolysin O, a thiol-activated cytolysin with 97.53% sequence identity, as the closest template. The Swiss model demonstrated superior validation compared to others. The protein folds into four functional domains characteristic of thiol-activated cytolysins: domain 3 likely forms beta-barrel channels, domains 1 and 2 are involved in oligomerization, and domain 4 recognizes cholesterol. Docking analysis revealed hydrophobic binding interactions with 8 of 11 residues in the cholesterol signature motif, including a hydrogen bond with Gly474 (2.29 Å). In conclusion, Bt produces a thermostable cholesterol-dependent cytolysin that shows strong similarity to anthrolysin O and perfringolysin O.
Correlation Between Prevalence of Some Colibactin Genes, Biofilm Formation, and Antimicrobial Resistance in Uropathogenic E. coli in Iraq Rukiya Saad Jassam, Husam Sabah Auhim Baghdad Science Journal, 2026 Colibactin is a genotoxin produced by Enterobacteriaceae via a polyketide synthase (pks) island cluster. There is less knowledge regarding the distribution of colibactin genes in E. coli isolates in Iraq and its correlation with biofilm and antibiotic susceptibility. Therefore, this study aimed to investigate the frequency of some colibactin genes (CIbA and CIbQ) in uropathogenic E. coli in Iraq and evaluate the correlation with biofilm and antimicrobial resistance. Between October 2023 and January 2024, 70 E. coli isolates were isolated from 120 females diagnosed with UTIs. Isolates were identified first by biochemical methods and confirmed molecularly by amplification of 16S rRNA gene with specific primers. PCR was employed to detect the CIbA and CIbQ genes. Biofilm production was assessed by the microtiter plates method, and antibiotic susceptibility was determined by the disc diffusion method. Results indicate a high prevalence of pks+ E. coli (40%, 28/70) compared to pks- E. coli (60%, 42/70) with non-significant differences (p > 0.05). E. coli produce different levels of biofilm with significant differences (p < 0.05). However, a statistically significant correlation has been found between E. coli (pks+) and a moderate biofilm formation with a significant difference (p < 0.05). pks+ E. coli isolates are more sensitive to all antibiotics under investigation, with a significant relation to fluoroquinolone group. Interestingly, current and previous results show that pks+ E. coli prevalence in Iraq has gradually increased over the last five years. High rates of pks-positive isolates in Iraqi urinary tract infections indicate the spread of highly genotoxic isolates.
Distribution and association of an usp genotoxin gene with biofilm formation in E. coli Husam Sabah Auhim, Hassan Majeed Rasheed Microbial Biosystems, 2025 Uropathogenic specific protein is a genotoxic protein targeting the DNA, leading to mutations and modifications in the normal cell's DNA and subsequently, cancer development. This study aims to determine the prevalence of the usp gene in Uropathogenic Escherichia coli isolated from females with urinary tract infections and study its correlation with biofilm formation. One hundred and five urine specimens were collected from female patients (20 to 55 years old) with urinary tract infections attending hospitals. Traditional laboratory methods using selective and differential culture media were used for initial bacterial isolation and identification, and molecular techniques that targeted a segment of the 16SrRNA gene with a specific primer pair were used to confirm the bacterial identification and usp gene detection using a conventional polymerase chain reaction. A microtiter plate method was used to assess the ability of isolates to produce biofilm. The bacterial isolation and identification results revealed (54.28%, 57/105) of isolates were Escherichia coli. The results of molecular detection of the usp gene revealed a considerable prevalence (98.2%, 56\57) in Uropathogenic Escherichia coli and a 100% ability to form a biofilm. The isolates exhibited different biofilm formation abilities, with a higher ability to form strong biofilm (42%, 24/57) followed by moderate and weak biofilm formation (35%,20/57) and (23%, 13/57), respectively. However, no statistical correlation between the usp gene and different abilities for biofilm formation has been found. The study’s limitation is that there is a small number of specimens due to the difficulty in specimen collection. In conclusion, the high prevalence of the usp gene in Uropathogenic Escherichia coli, although it does not correlate with biofilm, suggests its essential role in bacterial pathogenicity and the possibility of cancer disease in females with UTIs.
Molecular detection and the frequency of a pore-forming toxin in Enterococcus faecalis isolated from urinary tract infections Husam Sabah Auhim, Nisreen Hadi Odaa Microbes and Infectious Diseases, 2025 Background: Enterococcus faecalis is a causative agent for urinary tract infections (UTIs) in Iraq and worldwide, even though it is a commensal bacterium in human and animal intestines. It can cause different illnesses due to its ability to produce various virulence factors. A pore-forming toxin (cytolysin) is the most virulence factor in this bacterium. Objective: This study aims to molecularly investigate the frequency of cytolysin toxin among E. faecalis isolated from UTIs. Methods: A hundred and eighty urine specimens were collected from females diagnosed with UTIs. Traditional laboratory and molecular methods were used for bacterial identification and toxin detection using a modified DNA extraction method. Results: The findings revealed that 27.7% (50\180) of causative agents in UTIs were E. faecalis based on the molecular technique that targeted a housekeeping gene (ddI) with specific primers using polymerase chain reaction (PCR). Most of the isolates harboured the cytolysin toxin gene (cylLL) with a frequency rate of 92% (46\50). Conclusions: A considerable prevalence of cytolysin-positive isolates in UTIs, which is a worrying indicates of the extensive spreading of a toxic strain in UTIs. The modified method for DNA extraction in gene detection was successfully used to amplify a housekeeping gene (ddI) and a virulence gene (cylLL) for cytolysin toxin detection, and this approach can be utilised for rapid bacterial identification and gene detection in medical and research purposes with a large sample size in an inexpensive manner within a short time.
Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step Husam Sabah Auhim, B. Grigorenko, Tessa K. Harris, Ozan E. Aksakal, I. Polyakov, C. Berry, Gabriel Dos Passos Gomes, I. Alabugin, P. Rizkallah, A. Nemukhin, D. D. Jones Chemical Science, 2021 Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O2 located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O2 access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.
The Crystal Structure of Bacillus cereus HblL1 Harley L. Worthy, Lainey J. Williamson, Husam Sabah Auhim, Stephen H. Leppla, Inka Sastalla, D. Dafydd Jones, Pierre J. Rizkallah, Colin Berry Toxins, 2021 The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.
Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism Nehad Noby, Husam Sabah Auhim, Samuel Winter, Harley L. Worthy, Amira M. Embaby, Hesham Saeed, Ahmed Hussein, Christopher R. Pudney, Pierre J. Rizkallah, Stephen A. Wells, D. Dafydd Jones Open Biology, 2021 Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.
Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer Jacob R. Pope, Rachel L. Johnson, W. David Jamieson, Harley L. Worthy, Senthilkumar Kailasam, Rochelle D. Ahmed, Ismail Taban, Husam Sabah Auhim, Daniel W. Watkins, Pierre J. Rizkallah, Oliver K. Castell, D. Dafydd Jones Advanced Science, 2021 Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild‐type FPs are fused to a naturally occurring protein–protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction.
Positive functional synergy of structurally integrated artificial protein dimers assembled by Click chemistry Harley L. Worthy, Husam Sabah Auhim, W. David Jamieson, Jacob R. Pope, Aaron Wall, Robert Batchelor, Rachel L. Johnson, Daniel W. Watkins, Pierre Rizkallah, Oliver K. Castell, D. Dafydd Jones Communications Chemistry, 2019 Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.Oligomerisation is widely used to engineer proteins and peptides with desirable properties. Here, covalent homodimers and heterodimers of fluorescent proteins are designed in silico and experimentally shown to exhibit differing spectral properties depending upon the structure of the protein-protein interface.
