Toward Comprehensive Analysis of the 3D Chemistry of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are structured communities consisting of cells enmeshed in a self-generated extracellular matrix usually attached to a surface. They contain diverse classes of molecules including polysaccharides, lipids, proteins, nucleic acids, and diverse small organic molecules (primary and secondary metabolites) which are organized to optimize survival and facilitate dispersal to new colonization sites. In situ characterization of the chemical composition and structure of bacterial biofilms is necessary to fully understand their development on surfaces relevant to biofouling in health, industry, and the environment. Biofilm development has been extensively studied using confocal microscopy using targeted fluorescent labels providing important insights into the architecture of biofilms. Recently, cryopreparation has been used to undertake targeted in situ chemical characterization using Orbitrap secondary ion mass spectrometry (OrbiSIMS), providing a label-free method for imaging biofilms in their native state. Although the high mass resolution of OrbiSIMS enables more confident peak assignments, it is still very challenging to assign most of the peaks in the spectra due to complexity of SIMS spectra and lack of automatic peak assignment methods. Here, we analyze the same OrbiSIMS depth profile data generated from the frozen-hydrated biofilm, but employ a new untargeted chemical filtering process utilizing mass spectral databases to assign secondary ions to decipher the large number of fragments present in the SIMS spectra. To move towards comprehensive analysis of different chemistries in the sample, we apply a molecular formula prediction approach which putatively assigns 81% of peaks in the 3D OrbiSIMS depth profile analysis. This enables us to catalog over 1000 lipids and their fragments, 3500 protein fragments, 71 quorum sensing-related molecules (2-alkyl-4-quinolones and N-acylhomoserine lactones), 150 polysaccharide fragments, and glycolipids simultaneously from one data set and map these separated molecular classes spatially through a Pseudomonas aeruginosa biofilm. Assignment of different chemistries in this sample facilitates identification of differences between biofilms grown on biofilm-promoting and biofilm-resistant polymers.

SARS-CoV-2 variant biology: immune escape, transmission and fitness.

In late 2020, after circulating for almost a year in the human population, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited a major step change in its adaptation to humans. These highly mutated forms of SARS-CoV-2 had enhanced rates of transmission relative to previous variants and were termed 'variants of concern' (VOCs). Designated Alpha, Beta, Gamma, Delta and Omicron, the VOCs emerged independently from one another, and in turn each rapidly became dominant, regionally or globally, outcompeting previous variants. The success of each VOC relative to the previously dominant variant was enabled by altered intrinsic functional properties of the virus and, to various degrees, changes to virus antigenicity conferring the ability to evade a primed immune response. The increased virus fitness associated with VOCs is the result of a complex interplay of virus biology in the context of changing human immunity due to both vaccination and prior infection. In this Review, we summarize the literature on the relative transmissibility and antigenicity of SARS-CoV-2 variants, the role of mutations at the furin spike cleavage site and of non-spike proteins, the potential importance of recombination to virus success, and SARS-CoV-2 evolution in the context of T cells, innate immunity and population immunity. SARS-CoV-2 shows a complicated relationship among virus antigenicity, transmission and virulence, which has unpredictable implications for the future trajectory and disease burden of COVID-19.

Discovery of a polymer resistant to bacterial biofilm, swarming, and encrustation.

Innovative approaches to prevent catheter-associated urinary tract infections (CAUTIs) are urgently required. Here, we describe the discovery of an acrylate copolymer capable of resisting single- and multispecies bacterial biofilm formation, swarming, encrustation, and host protein deposition, which are major challenges associated with preventing CAUTIs. After screening ~400 acrylate polymers, poly(tert-butyl cyclohexyl acrylate) was selected for its biofilm- and encrustation-resistant properties. When combined with the swarming inhibitory poly(2-hydroxy-3-phenoxypropyl acrylate), the copolymer retained the bioinstructive properties of the respective homopolymers when challenged with Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Urinary tract catheterization causes the release of host proteins that are exploited by pathogens to colonize catheters. After preconditioning the copolymer with urine collected from patients before and after catheterization, reduced host fibrinogen deposition was observed, and resistance to diverse uropathogens was maintained. These data highlight the potential of the copolymer as a urinary catheter coating for preventing CAUTIs.

SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies.

Monoclonal antibodies (mAbs) offer a treatment option for individuals with severe COVID-19 and are especially important in high-risk individuals where vaccination is not an option. Given the importance of understanding the evolution of resistance to mAbs by SARS-CoV-2, we reviewed the available in vitro neutralization data for mAbs against live variants and viral constructs containing spike mutations of interest. Unfortunately, evasion of mAb-induced protection is being reported with new SARS-CoV-2 variants. The magnitude of neutralization reduction varied greatly among mAb-variant pairs. For example, sotrovimab retained its neutralization capacity against Omicron BA.1 but showed reduced efficacy against BA.2, BA.4 and BA.5, and BA.2.12.1. At present, only bebtelovimab has been reported to retain its efficacy against all SARS-CoV-2 variants considered here. Resistance to mAb neutralization was dominated by the action of epitope single amino acid substitutions in the spike protein. Although not all observed epitope mutations result in increased mAb evasion, amino acid substitutions at non-epitope positions and combinations of mutations also contribute to evasion of neutralization. This Review highlights the implications for the rational design of viral genomic surveillance and factors to consider for the development of novel mAb therapies.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

Tracking SARS-CoV-2 mutations and variants through the COG-UK-Mutation Explorer.

COG-UK Mutation Explorer (COG-UK-ME, https://sars2.cvr.gla.ac.uk/cog-uk/-last accessed date 16 March 2022) is a web resource that displays knowledge and analyses on SARS-CoV-2 virus genome mutations and variants circulating in the UK, with a focus on the observed amino acid replacements that have an antigenic role in the context of the human humoral and cellular immune response. This analysis is based on more than 2 million genome sequences (as of March 2022) for UK SARS-CoV-2 data held in the CLIMB-COVID centralised data environment. COG-UK-ME curates these data and displays analyses that are cross-referenced to experimental data collated from the primary literature. The aim is to track mutations of immunological importance that are accumulating in current variants of concern and variants of interest that could alter the neutralising activity of monoclonal antibodies (mAbs), convalescent sera, and vaccines. Changes in epitopes recognised by T cells, including those where reduced T cell binding has been demonstrated, are reported. Mutations that have been shown to confer SARS-CoV-2 resistance to antiviral drugs are also included. Using visualisation tools, COG-UK-ME also allows users to identify the emergence of variants carrying mutations that could decrease the neutralising activity of both mAbs present in therapeutic cocktails, e.g. Ronapreve. COG-UK-ME tracks changes in the frequency of combinations of mutations and brings together the curated literature on the impact of those mutations on various functional aspects of the virus and therapeutics. Given the unpredictable nature of SARS-CoV-2 as exemplified by yet another variant of concern, Omicron, continued surveillance of SARS-CoV-2 remains imperative to monitor virus evolution linked to the efficacy of therapeutics.

Fluid dynamics and cell-bound Psl polysaccharide allows microplastic capture, aggregation and subsequent sedimentation by Pseudomonas aeruginosa in water.

Decades after incorporating plastics into consumer markets, research shows that these polymers have spread worldwide. Fragmentation of large debris leads to smaller particles, collectively called microplastics (MPs), which have become ubiquitous in aquatic environments. A fundamental aspect of understanding the implications of MP contamination on ecosystems is resolving the complex interactions of these artificial substrates with microbial cells. Using polystyrene microparticles as model polymers, we conducted an exploratory study where these interactions are quantitatively analyzed using an in vitro system consisting of single-bacterial species capturing and aggregating MPs in water. Here we show that the production of Psl exopolysaccharide by Pseudomonas aeruginosa (PA) does not alter MPs colloidal stability but plays a key role in microspheres adhesion to the cell surface. Further aggregation of MPs by PA cells depends on bacterial mobility and the presence of sufficient flow to prevent rapid sedimentation of early MP-PA assembles. Surprisingly, cells in MP-PA aggregates are not in a sessile state despite the production of Psl, enhancing the motility of the aggregates by an order of magnitude relative to passive diffusion. The generated data could inform the creation of predictive models that accurately describe the dynamics and influence of bacterial growth on plastics debris.

Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7.

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.

SARS-CoV-2 variants, spike mutations and immune escape.

Although most mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome are expected to be either deleterious and swiftly purged or relatively neutral, a small proportion will affect functional properties and may alter infectivity, disease severity or interactions with host immunity. The emergence of SARS-CoV-2 in late 2019 was followed by a period of relative evolutionary stasis lasting about 11 months. Since late 2020, however, SARS-CoV-2 evolution has been characterized by the emergence of sets of mutations, in the context of 'variants of concern', that impact virus characteristics, including transmissibility and antigenicity, probably in response to the changing immune profile of the human population. There is emerging evidence of reduced neutralization of some SARS-CoV-2 variants by postvaccination serum; however, a greater understanding of correlates of protection is required to evaluate how this may impact vaccine effectiveness. Nonetheless, manufacturers are preparing platforms for a possible update of vaccine sequences, and it is crucial that surveillance of genetic and antigenic changes in the global virus population is done alongside experiments to elucidate the phenotypic impacts of mutations. In this Review, we summarize the literature on mutations of the SARS-CoV-2 spike protein, the primary antigen, focusing on their impacts on antigenicity and contextualizing them in the protein structure, and discuss them in the context of observed mutation frequencies in global sequence datasets.

Single-Cell Tracking on Polymer Microarrays Reveals the Impact of Surface Chemistry on Pseudomonas aeruginosa Twitching Speed and Biofilm Development.

Bacterial biofilms exhibit up to 1000 times greater resistance to antibiotic or host immune clearance than planktonic cells. Pseudomonas aeruginosa produces retractable type IV pili (T4P) that facilitate twitching motility on surfaces. The deployment of pili is one of the first responses of bacteria to surface interactions and because of their ability to contribute to cell surface adhesion and biofilm formation, this has relevance to medical device-associated infections. While polymer chemistry is known to influence biofilm development, its impact on twitching motility is not understood. Here, we combine a polymer microarray format with time-lapse automated microscopy to simultaneously assess P. aeruginosa twitching motility on 30 different methacrylate/acrylate polymers over 60 min post inoculation using a high-throughput system. During this critical initial period where the decision to form a biofilm is thought to occur, similar numbers of bacterial cells accumulate on each polymer. Twitching motility is observed on all polymers irrespective of their chemistry and physical surface properties, in contrast to the differential biofilm formation noted after 24 h of incubation. However, on the microarray polymers, P. aeruginosa cells twitch at significantly different speeds, ranging from 5 to ∼13 nm/s, associated with crawling or walking and are distinguishable from the different cell surface tilt angles observed. Chemometric analysis using partial least-squares (PLS) regression identifies correlations between surface chemistry, as measured by time-of-flight secondary ion mass spectrometry (ToF-SIMS), and both biofilm formation and single-cell twitching speed. The relationships between surface chemistry and these two responses are different for each process. There is no correlation between polymer surface stiffness and roughness as determined by atomic force measurement (AFM), or water contact angle (WCA), and twitching speed or biofilm formation. This reinforces the dominant and distinct contributions of material surface chemistry to twitching speed and biofilm formation.

Simultaneous Tracking of Pseudomonas aeruginosa Motility in Liquid and at the Solid-Liquid Interface Reveals Differential Roles for the Flagellar Stators.

Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a Pseudomonas aeruginosa wild-type strain and isogenic flagellar stator mutants (motAB and motCD) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the P. aeruginosa mot mutants, which decreased further on entering the near-surface environment. These results are consistent with the mot stators playing key roles in responding to the near-surface environment.IMPORTANCE We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of Pseudomonas aeruginosa, which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the mot stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.