Biology subjects

Gordon, V. D.

Publications and source records attributed to Gordon, V. D..

7 recordsLinked to original sources

bioRxiv2025

RNA Binding Proteins KhpA and KhpB Interact with Small Regulatory RNAs and Affect Global Gene Expression in Deinococcus radiodurans

Small regulatory RNAs (sRNAs) in bacteria often associate with RNA-binding proteins to gain intracellular stability and/or to enable regulatory efficiency. While much of the current knowledge about those sRNA binding proteins is derived from studies in Gram-negative organisms, the characterization of such proteins in Gram-positive species is still lagging behind. Here, we identified and characterized two sRNA binding proteins (KhpA and KhpB) in Deinococcus radiodurans, a Gram-positive bacterium that exhibits extreme resistance to radiation and other oxidative stressors. We demonstrate that KhpA and KhpB interact with key sRNAs in D. radiodurans and influence their stabilities. Although KhpA and KhpB interact with each other, they do not bind the sRNAs as a complex. Insightfully, KhpA and KhpB facilitate the interactions of the representative sRNAs PprS and Dsr9 in D. radiodurans with their respective mRNA targets, pprM and DR_1968. Through RNA-seq analysis, we further revealed that KhpA and KhpB have both overlapping and specific roles in a global gene regulation in D. radiodurans. Overall, this study expands our knowledge of posttranscriptional regulation in D. radiodurans and supports the growing consensus that KhpA and KhpB homologs constitute a new family of sRNA binding proteins in Gram-positive bacteria. IMPORTANCEThe bacterium Deinococcus radiodurans is the most radiation-resistant organism identified to date. Understanding the mechanisms of resistance of D. radiodurans is essential for leveraging this bacterium in biomedical and biomanufacturing applications. It was previously revealed that small regulatory RNAs (sRNAs) play crucial roles in the gene regulation of D. radiodurans. However, how these sRNAs are influenced by RNA binding proteins is poorly understood. Here we identified two conserved RNA binding proteins, KhpA and KhpB, as sRNA binding partners in D. radiodurans. These proteins affect the sRNA stability, sRNA-target interaction, and global gene regulation. Characterization of KhpA and KhpB will help us advance the understanding of how post-transcriptional network regulates the physiology and radioresistance of D. radiodurans.

molecular biology↗

bioRxiv2024

Surface remodeling and inversion of cell-matrix interactions underlies community recognition and dispersal in Vibrio cholerae biofilms

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. While it is commonly assumed that biofilm-dwelling cells are glued together by the matrix, how the cell-matrix interaction depends on the specific biochemistry of the matrix components and how this interaction varies during biofilm growth remains unclear. Here, we investigated cell-matrix interactions in Vibrio cholerae (Vc), the causative agent of cholera. We combine genetics, microscopy, simulation, and biochemical tools to show that Vc cells are not attractive to Vibrio polysaccharide (VPS), the main matrix component, but they can be bridged with each other and to the VPS network through crosslinking by Bap1. Downregulation of VPS and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results suggest a new conceptual model in understanding the intricate cell-matrix interaction as the major driver for biofilm development, which is potentially generalizable to certain other biofilm-forming species and exopolysaccharides.

biophysics↗

bioRxiv2023

Incorporation of collagen into Pseudomonas aeruginosa and Staphylococcus aureus biofilms impedes phagocytosis by neutrophils

Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS) and other components such as proteins. Matrix components can be produced by the microorganisms themselves but can also originate from the environment and then be incorporated into the biofilm. For example, we and our collaborators have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the matrices of Pseudomonas aeruginosa biofilms, altering biofilm mechanics. In an infection, the biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. P. aeruginosa, Staphylococcus aureus, and Burkholderia pseudomallei are human pathogens notable for forming biofilms in vitro and in vivo in tissues rich in collagen such as lung and skin. Here, we show that the incorporation of Type I collagen into P. aeruginosa, S. aureus, and B. pseudomallei biofilms significantly enhances biofilm elasticity and hinders phagocytosis of biofilm bacteria by human neutrophils. Additionally, enzymatic degradation of collagen using collagenase reverses these effects, increasing biofilm susceptibility to neutrophils. Our findings suggest that host materials play significant roles in stabilizing biofilms and may present promising targets for therapeutic interventions.

microbiology↗

bioRxiv2023

Structure-preserving fixation allows Scanning Electron Microscopy to reveal biofilm microstructure and interactions with immune cells

Pseudomonas aeruginosa is a pathogen that forms robust biofilms which are commonly associated with chronic infections and cannot be successfully cleared by the immune system. Neutrophils, the most common white blood cells, target infections with pathogen-killing mechanisms that are rendered largely ineffective by the protective physicochemical structure of a biofilm. Visualization of the complex interactions between immune cells and biofilms will advance understanding of how biofilms evade the immune system and could aid in developing treatment methods that promote immune clearance with minimal harm to the host. Scanning electron microscopy (SEM) distinguishes itself as a powerful, high-resolution tool for obtaining strikingly clear and detailed topographical images. However, taking full advantage of SEMs potential for high-resolution imaging requires that the fixation process simultaneously preserve both intricate biofilm architecture and the morphologies and structural signatures characterizing neutrophils responses at an infection site. Standard aldehyde-based fixation techniques result in significant loss of biofilm matrix material and morphologies of responding immune cells, thereby obscuring the details of immune interactions with the biofilm matrix. Here we show an improved fixation technique using the cationic dye alcian blue to preserve and visualize neutrophil interactions with the three-dimensional architecture of P. aeruginosa biofilms.

microbiology↗

bioRxiv2023

Physiological concentrations of calcium interact with alginate and extracellular DNA in the matrices of Pseudomonas aeruginosa biofilms to impede phagocytosis by neutrophils

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/563605v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@126dc6aorg.highwire.dtl.DTLVardef@50dab8org.highwire.dtl.DTLVardef@477fd6org.highwire.dtl.DTLVardef@19e8e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Suspended multiwalled, acid-functionalized carbon nanotubes promote aggregation of the opportunistic pathogen Pseudomonas aeruginosa

The increasing prevalence of carbon nanotubes (CNTs) as components of new functional materials has the unintended consequence of causing increases in CNT concentrations in aqueous environments. Aqueous systems are reservoirs for bacteria, including human and animal pathogens, that can form biofilms. At high concentrations, CNTs have been shown to display biocidal effects; however, at low concentrations, the interaction between CNTs and bacteria is more complicated, and antimicrobial action is highly dependent upon the properties of the CNTs in suspension. Here, impact of low concentrations of multiwalled CNTs (MWCNTs) on the biofilm-forming opportunistic human pathogen Pseudomonas aeruginosa is studied. Using phase contrast and confocal microscopy, flow cytometry, and antibiotic tolerance assays, it is found that sub-lethal concentrations (2 mg/L) of MWCNTs promote aggregation of P. aeruginosa into multicellular clusters. However, the antibiotic tolerance of these "young" bacterial-CNT aggregates is similar to that of CNT-free cultures. Overall, our results indicate that the co-occurrence of MWCNTs and P. aeruginosa in aqueous systems, which promotes the increased number and size of bacterial aggregates, could increase the dose to which humans or animals are exposed.

microbiology↗

bioRxiv2018

Increased production of the extracellular polysaccharide Psl can give a growth advantage to Pseudomonas aeruginosa in low-iron conditions

In infections, biofilm formation is associated with a number of fitness advantages, such as resistance to antibiotics and to clearance by the immune system. Biofilm formation has also been linked to fitness advantages in environments other than in vivo infections; primarily, biofilms are thought to help constituent organisms evade predation and to promote intercellular signaling. The opportunistic human pathogen Pseudomonas aeruginosa forms biofilm infections in lungs, wounds, and on implants and medical devices. However, the tendency toward biofilm formation originated in this bacteriums native environment, primarily plants and soil. Such environments are polymicrobial and often resource-limited. Other researchers have recently shown that the P. aeruginosa extracellular polysaccharide Psl can bind iron. For the lab strain PA01, Psl is also the dominant adhesive and cohesive \"glue\" holding together multicellular aggregates and biofilms. Here, we perform quantitative time-lapse confocal microscopy and image analysis of early biofilm growth by PA01. We find that aggregates of P. aeruginosa have a growth advantage over single cells of P. aeruginosa in the presence of Staphylococcus aureus in low-iron environments. Our results suggest the growth advantage of aggregates is linked to their high Psl content and to the production of an active factor by S. aureus. We posit that the ability of Psl to promote iron acquisition may have been linked to the evolutionary development of the strong biofilm-forming tendencies of P. aeruginosa.

microbiology↗