SACM - United Kingdom
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9667
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Item Restricted Investigation of Novel Type III Secretion System Protein Effectors of Aeromonas veronii(Saudi Digital Library, 2025) Alfaify, Abdulkhaleg; Shaw, JonathanThe type III secretion system (T3SS) plays a crucial role in the pathogenesis of various Gram-negative bacteria, enabling them to directly inject effector proteins into host cells. This study focuses on the identification, characterization, and functional analysis of putative T3SS effector proteins (AopX, PteB, and PteA) in Aeromonas veronii. Insertional gene mutants were generated and evaluated using the Galleria mellonella larvae infection model, showing significant alterations in virulence phenotypes. Transcriptomic analysis of an exsD mutant strain provided insights into the regulation of these novel effectors and demonstrated that they were part of theT3SS regulon. The study further characterized these proteins through expressing them in both Saccharomyces cerevisiae and A549 mammalian cells. Fluorescence imaging analysis showed that these proteins had significant impacts on host cell morphology and cytoskeletal organization. Additionally, BioID proximity labelling combined with mass spectrometry proteomics analysis identified host protein interactions and key cellular pathways that influenced by these bacterial effectors. Furthermore, protein purification was optimized using various chromatography approaches and computational modelling via AlphaFold and Phyre2 was used to predict and align protein structures. Overall we found that AopX, PteB, and PteA are regulated by the T3SS regulon. AopX primarily disrupts the actin cytoskeleton in both yeast and A549 cells, most probably through interacting with Filamin A, while PteB prevents bud formation and affects cell cycle progression by interacting with Serine/Threonine phosphatases. This comprehensive investigation enhances our understanding of T3SS effector proteins in A. veronii pathogenesis and provides insights into their regulation, structural features, and specific mechanisms of host cell manipulation.23 0Item Restricted Running the Gauntlet of the Bacterial Cell Wall(Newcastle University, 2025) Alofi, Amirah; Daniel, RichardIn nature, bacteria have a variety of shapes, from simple cocci and rods to more complicated spiral or appendage structures. In their life, they need a strong exoskeleton that can maintain their shape during growth and protect them from changes in environmental conditions. This rigid structure is generally provided by the cell wall and is considered an essential component in the bacterial cell. However, the cell wall represents a complex logistical problem in terms of the controlled synthesis and degradation that is required to maintain shape, permit enlargement, and prevent lysis. Significant advances have been made in understanding wall synthesis, and in Gram-negative bacteria synthesis can be integrated with degradation. But, for Gram-positive bacteria the regulation of cell wall degradation is poorly characterised. Genetically the genes encoding the key enzymes are known and the expression of these enzymes are known to be highly regulated. However, understanding how the biochemical activity of these enzymes is restricted and regulated, presumably within the cell wall or on the outside surface of the cell is not clear. The results obtained in this laboratory have indicated that altered cell wall composition or media components modulate the level of the hydrolysis enzyme activity in some way during vegetive growth. This study aimed to extend our understanding of the autolytic systems and how the enzymatic activity may be regulated by components of the cell envelope or minor modifications of the cell wall material.20 0Item Restricted Oral biofilm and host-pathogen models: a semi- systematic review and future perspectives(University Of Glasgow, 2024-08) Alshehri, Khalid; Brown, JasonAbstract Introduction: Oral biofilms, complex microbial communities found on various surfaces within the oral cavity, play a critical role in the development and progression of oral diseases such as dental caries, periodontal diseases, and mucosal infections. Understanding the formation, structure, and pathogenicity of these biofilms is essential for improving prevention and treatment strategies. Aims: This review aims to evaluate recent advancements in the development and application of in vitro multi-species oral biofilm models, with a focus on studies published between January 2019 and July 2024. The review seeks to identify gaps in current research and suggest future directions for enhancing the physiological relevance of these models. Methods: A systematic literature search was conducted in the PubMed database, following PRISMA guidelines. Studies were selected based on predefined inclusion and exclusion criteria, focusing on multi-species biofilm models in vitro. The review analyzed methodologies, findings, and limitations of the selected studies. Findings: The review identified six key studies employing various in vitro models, ranging from continuous flow systems to static models. These studies highlighted the importance of specific microbial interactions, biofilm maturation processes, and the impact of different substrates on biofilm formation. However, limitations were noted in replicating the complexity of the in vivo oral environment, particularly in capturing the dynamic conditions and microbial diversity. Discussion: While significant progress has been made in the development of in vitro biofilm models, challenges remain in creating systems that accurately mimic the oral microenvironment. Advances in microfluidic devices and 'OMICs' technologies offer promising avenues for future research. Additionally, there is a need for long-term studies that better reflect the chronic nature of biofilm-related infections. Conclusion: The development of in vitro models that closely replicate the in vivo conditions of the oral cavity is crucial for advancing our understanding of oral biofilms and their role in disease progression. Future research should focus on integrating advanced technologies and improving model complexity to enhance the predictive value of these systems for clinical applications.15 0Item Restricted Epigenetic Gene Regulation by the Type I Restriction Modification Systems(University of Leicester, 2024-04) Althari, Yasmeen; Oggioni, MarcoEpigenetic modifications mediated by type I restriction modification systems in prokaryotes have been linked to gene regulation. In Streptococcus pneumoniae, differential expression of alternative specificity subunits within the SpnIII type I restriction modification system had been shown to selectively modulate bacterial virulence. Given the presence of this system in the core genome of pneumococci, but not of the related Streptococcus mitis, SpnIII could represent a conserved phase-variable regulatory mechanism operating on a global scale. However, this phenomenon had yet to be examined at the single-gene level, and the underlying molecular mechanism remained unexplored. In this study, I utilised pneumococcal strains locked for alternative specificity subunits (no phase-variation at the locus) to validate methylation-dependent differential gene expression of several model genes using various transcriptomic and translation reporter assays. Additionally, I delved into the complexity of recombination within the spnIII locus, which is partially governed by a site-specific tyrosine recombinase whose mechanism of controlling the rate of recombination remains elusive. To investigate the regulation of this recombinase, I explored the possibility of its control by a hairpin structure in the 5-prime UTR of the gene representing a potential RNA thermosensor, considering the recognised temperature sensitivity of recombination. To provide evidence to support my hypothesis on epigenetic gene regulation, I analysed the non-phase variable prototype type I EcoKI system in Escherichia coli which revealed a similar methylation-dependent differential expression following deletion of the EcoKI methyltransferase. This discovery underscores the epigenetic impact of type I RMSs, suggesting a widespread occurrence and possibly a global relevance of this phenomenon across bacterial genera. Overall, my findings propose that methylation influences both local gene topology and global genome architecture, thus playing a crucial role in methylation-mediated regulation. This mechanism involves the interplay between methylation and DNA-binding proteins, which collectively shape the overall genome architecture and transcriptional landscape.14 0Item Restricted Comparison of long-read and short-read bacterial DNA sequencing(King's College London, 2024-08-30) Alqirnas, Mohammed; Carpenter, Guy; Cleaver, LeanneThis study aimed to compare long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies for characterizing the diversity and composition of in vitro oral biofilms. An oral biofilm model was established using hydroxyapatite discs to mimic the tooth surface. Saliva samples from six healthy participants were pooled and used to inoculate the discs, which underwent aerobic and anaerobic incubation phases. Biofilm formation was assessed using confocal microscopy with LIVE/DEAD staining, revealing heterogeneous growth patterns with coverage ranging from 17% to 47%. DNA extraction was carried out using the GenElute Bacterial Genomic DNA Kit, with yields showing significant variations across experiments (0-5 ng/μL). Interestingly, gel electrophoresis showed no difference in DNA fragment lengths between samples prepared for short-read and long-read sequencing. The experiment also highlighted the potential benefits of CO2-rich environments for early colonizer growth, particularly Streptococcus species. While the biofilm model was successfully established, the results underline the need for protocol optimization, particularly in DNA extraction and biofilm cultivation. This research provides insights into the complexity of oral microbiome analysis and sets the stage for future comparative studies on advanced sequencing technologies in oral microbiome. The findings also emphasize the importance of refining the in vitro experimental protocol, from biofilm cultivation to DNA sequencing and data analysis.13 0Item Restricted Comparison of long-read and short-read bacterial DNA sequencing(King's College London, 2024-08) Alqirnas, Mohammed; Carpenter, Guy; Cleaver, LeanneThis study aimed to compare long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies for characterizing the diversity and composition of in vitro oral biofilms. An oral biofilm model was established using hydroxyapatite discs to mimic the tooth surface. Saliva samples from six healthy participants were pooled and used to inoculate the discs, which underwent aerobic and anaerobic incubation phases. Biofilm formation was assessed using confocal microscopy with LIVE/DEAD staining, revealing heterogeneous growth patterns with coverage ranging from 17% to 47%. DNA extraction was carried out using the GenElute Bacterial Genomic DNA Kit, with yields showing significant variations across experiments (0-5 ng/μL). Interestingly, gel electrophoresis showed no difference in DNA fragment lengths between samples prepared for short-read and long-read sequencing. The experiment also highlighted the potential benefits of CO2-rich environments for early colonizer growth, particularly Streptococcus species. While the biofilm model was successfully established, the results underline the need for protocol optimization, particularly in DNA extraction and biofilm cultivation. This research provides insights into the complexity of oral microbiome analysis and sets the stage for future comparative studies on advanced sequencing technologies in oral microbiome. The findings also emphasize the importance of refining the in vitro experimental protocol, from biofilm cultivation to DNA sequencing and data analysis.29 0Item Restricted The role of commensal bacteria in the maintenance of the tight junction skin barrier(Saudi Digital Library, 2023) Aldehalan, Faye; Catherine, O'neillThe skin is a critical barrier that prevents water and heat loss from the body and prevents ingress of toxins and pathogens. This barrier comprises the physical (stratum corneum and tight junctions (TJs)), chemical and immune components. Recent studies have shown that the skin microbiota also plays a role in epidermal barrier function by preventing infection by pathogenic bacteria and enhancing skin immune responses. Some recent studies have also suggested that the physical barrier can be regulated by bacterial metabolites such as butyrate, from the fermentation of glycerol. However, to date most studies have been conducted in mouse models using consortia of bacteria and few studies have looked at individual bacteria and their effects on the barrier. The aim of this doctoral thesis was to identify human skin bacterial isolates that have the potential to regulate TJ barrier function possibly via production of butyrate and other metabolites. Skin swabs were collected from five healthy participants to create a biobank of skin commensal bacteria using culture-based methods. The isolated species were used in screening assays of TJ function in primary normal human epidermal keratinocytes (NHEKs). One of the enhancers of the intestinal barrier is butyrate, a bacterial metabolite from fermentation of carbohydrates. Similar mechanisms may also exist in the skin since some skin commensal bacteria have been shown to produce butyrate from the fermentation of glycerol. A significant increase in transepithelial electrical resistance (TEER) was observed in NHEKs treated with 4 mM butyrate for 24 h compared to untreated cells. However, treatment with lower concentrations (2 and 0.5 mM) induced no change in TEER. Staphylococcal isolates were able to ferment glycerol as a source of carbohydrate, but the concentrations of butyrate produced were low. In keeping with this, the glycerol-fermented supernatants of all of the tested species (S. epidermidis, S. hominis, S. capitis S. lugdunensis, and S. caprae) had no impact on TJ barrier function in NHEKs. The effect of cell-free supernatants of different skin commensal isolates grown in tryptic soy broth (to maximise the production of metabolites), on TJ integrity were investigated in NHEKs. The supernatants from S. hominis, S. aureus and M. luteus significantly increased the TJ barrier integrity as measured by TEER. However, the supernatants of S. epidermidis, S. capitis S. lugdunensis, and S. caprae had no impact on TJs. Metabolite identification by mass spectrometry showed that all the species produced multiple metabolites, with many in common between the different isolates. However, tryptophol, a known enhancer of the intestinal TJ barrier was detected only in the supernatants of the TJ barrier-enhancing species. There was almost zero production by the ineffective species. Further investigation sought to determine whether this was a general effect of a certain species, or whether there were any strain-specific effects. Therefore, other isolates of S. hominis were investigated for their ability to increase TJ function. Only specific isolates of S. hominis could regulate TJ function in NHEKs and this correlated with the production of tryptophol. Furthermore, an isolate that was unable to produce tryptophol or modify TJ function, was sequenced and found to contain mutations in a gene for tryptophol production. This study shows for the first time that specific bacteria within the skin microbiota can produce metabolites with the capacity to regulate human keratinocytes tight junction function. The identification of such activities could be utilised in the development of therapeutic approaches to different skin conditions where the barrier is known to be aberrant.10 0