Biology subjects

Whitchurch, C. B.

Publications and source records attributed to Whitchurch, C. B..

9 recordsLinked to original sources

bioRxiv2025

Tissue destruction during food spoilage is associated with the formation of biofilms by Pseudomonas species

Members of the Pseudomonas genus are common spoilers of a range of meat, dairy and vegetable products. While we have a good understanding of the Pseudomonas species typically responsible for spoilage, we know very little about how these bacteria interact with food surfaces during spoilage. Here we assessed the spoilage capabilities of a large panel (n=124) of Pseudomonas species food-derived isolates on meat (chicken) and leafy greens (spinach). Most isolates (71/124) were capable of spoiling both foods, but some were only capable of spoiling only chicken (21/124) or spinach (23/124), or neither (9/124). Our data also demonstrated that the type of fresh food the strain was isolated from influenced spoilage capabilities: strains isolated from meat were likely to spoil both chicken and spinach, while those isolated from leafy greens were more likely to spoil only spinach. We used fluorescence microscopy to visualise how Pseudomonas spoilage species interacted with the meat or leaf tissue and observed significant tissue destruction associated with biofilm formation. For chicken, this was associated with the formation of dense biofilm pillars that penetrated deep into the tissue. For spinach we observed biofilms on the leaf in areas of tissue degradation. Finally, we explored the correlation between potentially relevant phenotypes (in vitro biofilm, motility and secreted enzyme production) and spoilage capabilities. There was no significant correlation between any of these phenotypes and spoilage, except for secreted protease activity and chicken spoilage. Overall, this study increases our understanding of processes involved in food spoilage by Pseudomonas species.

microbiology↗

bioRxiv2024

FakA impacts antiseptic susceptibility in Staphylococcus aureus and Enterococcus faecalis

Biocides are widely used to control and prevent healthcare-associated infections and understanding how nosocomial pathogens respond to biocidal agents is key to improving infection prevention and control products and practices. An evolution model was used to study how Staphylococcus aureus and Enterococcus faecalis responded after repeated exposure to sub-lethal concentrations of chlorhexidine digluconate (CHX) and octenidine dihydrochloride (OCT) when grown planktonically and as biofilms. Both pathogens were able to adapt to grow at concentrations above the MIC of both biocides with planktonic lineages surviving at higher concentrations of both agents than biofilm lineages. Exposure to CHX was linked with lower biofilm biomass production in E. faecalis although biofilm biomass increased for S. aureus isolates after exposure to both agents. Evolved isolates had no major fitness deficit and only low-level changes to susceptibility to antibiotics were observed after biocide exposure. Sequencing of biocide adapted mutants repeatedly identified mutations within fakA encoding a fatty acid kinase in independent lineages of S. aureus after exposure to both biocides in all conditions. Analogous changes were observed within the homologous gene in parallel experiments with Enterococcus faecalis. Further assays to study the mechanistic basis and relationship to phospholipid production showed that evolved isolates with fakA mutations accumulated less ethidium bromide than parent strains, exhibited altered cell envelope morphology and decreased susceptibility to daptomycin. This data shows important pathogens can evolve limited tolerance to two common biocides but that this has collateral impacts on biofilm formation, colony morphology and fitness. FakA appears to play an important role in biocide tolerance.

microbiology↗

bioRxiv2023

Effect of caffeine and other xanthines on liver sinusoidal endothelial cell ultrastructure

Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as food additives. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 g/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 g/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery.

cell biology↗

bioRxiv2020

Bacteriophage infection produces membrane vesicles in Escherichia coli via both explosive cell lysis and membrane blebbing

Membrane Vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV biogenesis in Pseudomonas aeruginosa that involves explosive cell lysis events which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV biogenesis is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of E. coli cells infected with either bacteriophage T4 or T7, produced MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV biogenesis. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

microbiology↗

bioRxiv2020

Multiple Holins Contribute to Extracellular DNA Release in Pseudomonas aeruginosa Biofilms

Bacterial biofilms are comprised of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a glue, facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

microbiology↗

bioRxiv2020

Characterising the mechanism of action of an ancient antimicrobial, honey, using modern transcriptomics

Manuka honey has broad-spectrum antimicrobial activity and unlike traditional antibiotics, resistance to its killing effects has not been reported. However, its mechanism of action remains unclear. Here we investigated the mechanism of action of manuka honey and its key antibacterial components using a transcriptomic approach in a model organism, Pseudomonas aeruginosa. We show that no single component of honey can account for its total antimicrobial action, and that honey affects the expression of genes in the SOS response, oxidative damage and quorum sensing. Manuka honey uniquely affects genes involved in the explosive cell lysis process and in maintaining the electron transport chain, causing protons to leak across membranes and collapsing the proton motive force; and induces membrane depolarisation and permeabilisation in P. aeruginosa. These data indicate that the activity of manuka honey comes from multiple mechanisms of action that do not engender bacterial resistance. ImportanceThe threat of antimicrobial resistance to human health has prompted interest in complex, natural products with antimicrobial activity. Honey has been an effective topical wound treatment throughout history, predominantly due to its broad-spectrum antimicrobial activity. Unlike traditional antibiotics, honey-resistant bacteria have not been reported, however, honey remains underutilised in the clinic in part due to a lack of understanding of its mechanism of action. Here we demonstrate that honey affects multiple processes in bacteria, and this is not explained by its major antibacterial components. Honey also uniquely affects bacterial membranes and this can be exploited for combination therapy with antibiotics that are otherwise ineffective on their own. We argue that honey should be included as part of the current array of wound treatments due to its effective antibacterial activity that does not promote resistance in bacteria.

microbiology↗

bioRxiv2019

Pseudomonas aeruginosa is capable of natural transformation in biofilms

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologs of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa.

microbiology↗

bioRxiv2019

ChpC controls twitching motility-mediated expansion of Pseudomonas aeruginosa biofilms in response to serum albumin, mucin and oligopeptides

Twitching motility-mediated biofilm expansion occurs via coordinated, multi-cellular collective behaviour to allow bacteria to actively expand across surfaces. Type-IV pili (T4P) are cell-associated virulence factors which mediate this expansion via rounds of extension, surface attachment and retraction. The Chp chemosensory system is thought to respond to environmental signals to regulate the biogenesis, assembly and twitching motility function of T4P. In other well characterised chemosensory systems, methyl-accepting chemotaxis proteins (MCPs) feed environmental signals through a CheW adapter protein to the histidine kinase CheA to modulate motility. The Pseudomonas aeruginosa Chp system has two CheW adapter proteins, PilI and ChpC, and an MCP PilJ that likely interacts via PilI with the histidine kinase ChpA. It is thought that ChpC associates with other MCPs to feed environmental signals into the system, however no such signals have been identified. In the current study we show that ChpC is involved in the response to host-derived signals serum albumin, mucin and oligopeptides. We demonstrate that these signals stimulate an increase in twitching motility, as well as in levels of 3-5-cyclic adenosine monophosphate (cAMP) and surface-assembled T4P. Interestingly, our data shows that changes in cAMP and surface piliation levels are independent of ChpC but that the twitching motility response to these environmental signals requires ChpC. Based upon our data we propose a model whereby ChpC associates with an MCP other than PilJ to feed these environmental signals through the Chp system to control twitching motility. The MCP PilJ and the CheW adapter PilI then modulate T4P surface levels to allow the cell to continue to undergo twitching motility. Our study is the first to link environmental signals to the Chp chemosensory system and refines our understanding of how this system controls twitching motility-mediated biofilm expansion in P. aeruginosa.

microbiology↗

bioRxiv2018

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggests a differential effect on twitching motility by flagellum components based upon their cellular location. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

genomics↗