High-Performance Dopamine-Based Supramolecular Bio-Adhesives.

The need for wound closure or surgical procedures has been commonly met by the application of sutures. Unfortunately, these are often invasive or subject to contamination. Alternative solutions are offered by surgical adhesives that can be applied and set without major disruption; a new class of supramolecular-based adhesives provides potential solutions to some of these challenges. In this study, a series of polymers utilizing dopamine as a self-assembling unit are synthesized. It is found that these motifs act as extremely effective adhesives, with control over the mechanical strength of the adhesion and materials' tensile properties enabled by changing monomer feed ratios and levels of cross-linking. These materials significantly outperform commercially available bio-adhesives, showing yield strengths after adhesion at least two times higher than that of BioGlue and Tisseel, as well as the ability to re-adhere with significant recovery of adhesion strength. Promisingly, the materials are shown to be non-cytotoxic, with cell viability > 90%, and able to perform in aqueous environments without significant loss in strength. Finally, the removal of the materials, is possible using benign organic solvents such as ethanol. These properties all demonstrate the effectiveness of the materials as potential bio-adhesives, with potential advantages for use in surgery.

Mucin Transiently Sustains Coronavirus Infectivity through Heterogenous Changes in Phase Morphology of Evaporating Aerosol.

Respiratory pathogens can be spread though the transmission of aerosolised expiratory secretions in the form of droplets or particulates. Understanding the fundamental aerosol parameters that govern how such pathogens survive whilst airborne is essential to understanding and developing methods of restricting their dissemination. Pathogen viability measurements made using Controlled Electrodynamic Levitation and Extraction of Bioaerosol onto Substrate (CELEBS) in tandem with a comparative kinetics electrodynamic balance (CKEDB) measurements allow for a direct comparison between viral viability and evaporation kinetics of the aerosol with a time resolution of seconds. Here, we report the airborne survival of mouse hepatitis virus (MHV) and determine a comparable loss of infectivity in the aerosol phase to our previous observations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the addition of clinically relevant concentrations of mucin to the bioaerosol, there is a transient mitigation of the loss of viral infectivity at 40% RH. Increased concentrations of mucin promoted heterogenous phase change during aerosol evaporation, characterised as the formation of inclusions within the host droplet. This research demonstrates the role of mucus in the aerosol phase and its influence on short-term airborne viral stability.

Direct visualization of sequence-specific DNA binding by gonococcal type IV pili.

Neisseria gonorrhoeae, the causative agent of gonorrhoea, is a major burden on global healthcare systems, with an estimated ~80-90 million new global cases annually. This burden is exacerbated by increasing levels of antimicrobial resistance, which has greatly limited viable antimicrobial therapies. Decreasing gonococcal drug susceptibility has been driven largely by accumulation of chromosomal resistance determinants, which can be acquired through natural transformation, whereby DNA in the extracellular milieu is imported into cells and incorporated into the genome by homologous recombination. N. gonorrhoeae possesses a specialized system for DNA uptake, which strongly biases transformation in favour of DNA from closely related bacteria by recognizing a 10-12 bp DNA uptake sequence (DUS) motif, which is highly overrepresented in their chromosomal DNA. This process relies on numerous proteins, including the DUS-specific receptor ComP, which assemble retractile protein filaments termed type IV pili (T4P) extending from the cell surface, and one model for neisserial DNA uptake proposes that these filaments bind DNA in a DUS-dependent manner before retracting to transport DNA into the periplasm. However, conflicting evidence indicates that elongated pilus filaments may not have such a direct role in DNA binding uptake as this model suggests. Here, we quantitatively measured DNA binding to gonococcal T4P fibres by directly visualizing binding complexes with confocal fluorescence microscopy in order to confirm the sequence-specific, comP-dependent DNA binding capacity of elongated T4P fibres. This supports the idea that pilus filaments could be responsible for initially capturing DNA in the first step of sequence-specific DNA uptake.

Rapid Detection of Neisseria gonorrhoeae Genomic DNA Using Gold Nanoprobes Which Target the Gonococcal DNA Uptake Sequence.

The rapid spread of antimicrobial resistant Neisseria gonorrhoeae continues to pose a serious threat to global health. To successfully treat and control gonococcal infections, rapid diagnosis is critical. Currently, nucleic acid amplification tests are the recommended diagnostic, however, these are both technically demanding and time consuming, making them unsuitable for resource-poor clinics. Consequently, there is a substantial need for an affordable, point-of-care diagnostic to use in these settings. In this study, DNA-functionalised gold nanoparticles (gold nanoprobes), with the ability to specifically detect the DNA Uptake Sequence (DUS) of Neisseria gonorrhoeae, were prepared. Using complementary annealing, the gold nanoprobes were shown to hybridise to genomic gonococcal DNA, causing a significant shift in their salt stability. By exploiting the shift in nanoprobe stability under the presence of target DNA, a solution-based colorimetric diagnostic for gonococcal DNA was prepared. Detection of purified genomic DNA was achieved in under 30 minutes, with a detection limit of 15.0 ng. Significantly, testing with DNA extracted from an off-target control organism suggested specificity for Neisseria. These results highlight the potential of DUS-specific gold nanoprobes in the rapid point-of-care diagnosis of gonococcal infections.

The dynamics of SARS-CoV-2 infectivity with changes in aerosol microenvironment.

Understanding the factors that influence the airborne survival of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARS-CoV-2 in aerosol droplets (∼5 to 10 µm equilibrated radius) over timescales spanning 5 s to 20 min using an instrument to probe survival in a small population of droplets (typically 5 to 10) containing ∼1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to ∼10% of the starting value was observable for SARS-CoV-2 over 20 min, with a large proportion of the loss occurring within the first 5 min after aerosolization. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallize at relative humidities (RHs) below 50%, leading to a near-instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH, the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 min, beyond which it decays to only 10% remaining infectious after 10 min. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilization of CO2 from bicarbonate buffer within the droplet. Four different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.

Identification of integrative and conjugative elements in pathogenic and commensal Neisseriaceae species via genomic distributions of DNA uptake sequence dialects.

Mobile genetic elements (MGEs) are key factors responsible for dissemination of virulence determinants and antimicrobial-resistance genes amongst pathogenic bacteria. Conjugative MGEs are notable for their high gene loads donated per transfer event, broad host ranges and phylogenetic ubiquity amongst prokaryotes, with the subclass of chromosomally inserted integrative and conjugative elements (ICEs) being particularly abundant. The focus on a small number of model systems has biased the study of ICEs towards those conferring readily selectable phenotypes to host cells, whereas the identification and characterization of integrated cryptic elements remains challenging. Even though antimicrobial resistance and horizontally acquired virulence genes are major factors aggravating neisserial infection, conjugative MGEs of Neisseria gonorrhoeae and Neisseria meningitidis remain poorly characterized. Using a phenotype-independent approach based on atypical distributions of DNA uptake sequences (DUSs) in MGEs relative to the chromosomal background, we have identified two groups of chromosomally integrated conjugative elements in Neisseria: one found almost exclusively in pathogenic species possibly deriving from the genus Kingella, the other belonging to a group of Neisseria mucosa-like commensals. The former element appears to enable transfer of traditionally gonococcal-specific loci such as the virulence-associated toxin-antitoxin system fitAB to N. meningitidis chromosomes, whilst the circular form of the latter possesses a unique attachment site (attP) sequence seemingly adapted to exploit DUS motifs as chromosomal integration sites. In addition to validating the use of DUS distributions in Neisseriaceae MGE identification, the >170 identified ICE sequences provide a valuable resource for future studies of ICE evolution and host adaptation.

Fusobacterium spp. target human CEACAM1 via the trimeric autotransporter adhesin CbpF.

Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis are pathogenic bacteria adapted to reside on human respiratory mucosal epithelia. One common feature of these species is their ability to target members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, especially CEACAM1, which is achieved via structurally distinct ligands expressed by each species. Beside respiratory epithelial cells, cells at the dentogingival junction express high levels of CEACAM1. It is possible that bacterial species resident within the oral cavity also utilise CEACAM1 for colonisation and invasion of gingival tissues. From a screen of 59 isolates from the human oral cavity representing 49 bacterial species, we identified strains from Fusobacterium bound to CEACAM1. Of the Fusobacterium species tested, the CEACAM1-binding property was exhibited by F. nucleatum (Fn) and F. vincentii (Fv) but not F. polymorphum (Fp) or F. animalis (Fa) strains tested. These studies identified that CEACAM adhesion was mediated using a trimeric autotransporter adhesin (TAA) for which no function has thus far been defined. We therefore propose the name CEACAM binding protein of Fusobacterium (CbpF). CbpF was identified to be present in the majority of unspeciated Fusobacterium isolates confirming a subset of Fusobacterium spp. are able to target human CEACAM1.

Bioinformatic analysis of meningococcal Msf and Opc to inform vaccine antigen design.

Neisseria meningitidis is an antigenically and genetically variable Gram-negative bacterium and a causative agent of meningococcal meningitis and septicaemia. Meningococci encode many outer membrane proteins, including Opa, Opc, Msf, fHbp and NadA, identified as being involved in colonisation of the host and evasion of the immune response. Although vaccines are available for the prevention of some types of meningococcal disease, none currently offer universal protection. We have used sequences within the Neisseria PubMLST database to determine the variability of msf and opc in 6,500 isolates. In-silico analysis revealed that although opc is highly conserved, it is not present in all isolates, with most isolates in clonal complex ST-11 lacking a functional opc. In comparison, msf is found in all meningococcal isolates, and displays diversity in the N-terminal domain. We identified 20 distinct Msf sequence variants (Msf SV), associated with differences in number of residues within the putative Vn binding motifs. Moreover, we showed distinct correlations with certain Msf SVs and isolates associated with either hyperinvasive lineages or those clonal complexes associated with a carriage state. We have demonstrated differences in Vn binding between three Msf SVs and generated a cross reactive Msf polyclonal antibody. Our study has highlighted the importance of using large datasets to inform vaccine development and provide further information on the antigenic diversity exhibited by N. meningitidis.

Helicobacter pylori adhesin HopQ engages in a virulence-enhancing interaction with human CEACAMs.

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a ß-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.

Comparative Genomic Analyses of the Moraxella catarrhalis Serosensitive and Seroresistant Lineages Demonstrate Their Independent Evolution.

The bacterial speciesMoraxella catarrhalishas been hypothesized as being composed of two distinct lineages (referred to as the seroresistant [SR] and serosensitive [SS]) with separate evolutionary histories based on several molecular typing methods, whereas 16S ribotyping has suggested an additional split within the SS lineage. Previously, we characterized whole-genome sequences of 12 SR-lineage isolates, which revealed a relatively small supragenome when compared with other opportunistic nasopharyngeal pathogens, suggestive of a relatively short evolutionary history. Here, we performed whole-genome sequencing on 18 strains from both ribotypes of the SS lineage, an additional SR strain, as well as four previously identified highly divergent strains based on multilocus sequence typing analyses. All 35 strains were subjected to a battery of comparative genomic analyses which clearly show that there are three lineages-the SR, SS, and the divergent. The SR and SS lineages are closely related, but distinct from each other based on three different methods of comparison: Allelic differences observed among core genes; possession of lineage-specific sets of core and distributed genes; and by an alignment of concatenated core sequences irrespective of gene annotation. All these methods show that the SS lineage has much longer interstrain branches than the SR lineage indicating that this lineage has likely been evolving either longer or faster than the SR lineage. There is evidence of extensive horizontal gene transfer (HGT) within both of these lineages, and to a lesser degree between them. In particular, we identified very high rates of HGT between these two lineages for ß-lactamase genes. The four divergent strains aresui generis, being much more distantly related to both the SR and SS groups than these other two groups are to each other. Based on average nucleotide identities, gene content, GC content, and genome size, this group could be considered as a separate taxonomic group. The SR and SS lineages, although distinct, clearly form a single species based on multiple criteria including a large common core genome, average nucleotide identity values, GC content, and genome size. Although neither of these lineages arose from within the other based on phylogenetic analyses, the question of how and when these lineages split and then subsequently reunited in the human nasopharynx is explored.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Moraxella catarrhalis adhesin UspA1-derived recombinant fragment rD-7 induces monocyte differentiation to CD14+CD206+ phenotype.

Circulating monocytes in the bloodstream typically migrate to other tissues and differentiate into tissue resident macrophages, the process being determined by the constituents of the microenvironments encountered. These may include microbes and their products. In this study, we investigated whether Moraxella catarrhalis Ubiquitous Surface Protein A1 (UspA1), known to bind to a widely expressed human cell surface receptor CEACAM1, influences monocyte differentiation as receptor engagement has been shown to have profound effects on monocytes. We used the recombinant molecules corresponding to the regions of UspA1 which either bind (rD-7; UspA1527-665) or do not bind (r6-8; UspA1659-863) to CEACAM1 and investigated their effects on CD206, CD80 and CD86 expression on freshly isolated human CD14+ monocytes from peripheral blood mononuclear cells (PBMC). Exposure to rD-7, but not r6-8, biased monocyte differentiation towards a CD14+CD206+ phenotype, with reduced CD80 expression. Monocytes treated with rD-7 also secreted high levels of IL-1ra and chemokine IL-8 but not IL-10 or IL-12p70. The effects of rD-7 were independent of any residual endotoxin. Unexpectedly, these effects of rD-7 were also independent of its ability to bind to CEACAM1, as monocyte pre-treatment with the anti-CEACAM antibody A0115 known to inhibit rD-7 binding to the receptor, did not affect rD-7-driven differentiation. Further, another control protein rD-7/D (a mutant form of rD-7, known not to bind to CEACAMs), also behaved as the parent molecule. Our data suggest that specific regions of M. catarrhalis adhesin UspA1 may modulate inflammation during infection through a yet unknown receptor on monocytes.

A novel group of Moraxella catarrhalis UspA proteins mediates cellular adhesion via CEACAMs and vitronectin.

Moraxella catarrhalis (Mx) is a common cause of otitis media and exacerbation of chronic obstructive pulmonary disease, an increasing worldwide problem. Surface proteins UspA1 and UspA2 of Mx bind to a number of human receptors and may function in pathogenesis. Genetic recombination events in the pathogen can generate hybrid proteins termed UspA2H. However, whether certain key functions (e.g. UspA1-specific CEACAM binding) can be exchanged between these adhesin families remains unknown. In this study, we have shown that Mx can incorporate the UspA1 CEACAM1-binding region not only into rare UspA1 proteins devoid of CEACAM-binding ability, but also into UspA2 which normally lack this capacity. Further, a screen of Mx isolates revealed the presence of novel UspA2 Variant proteins (UspA2V) in ∼14% of the CEACAM-binding population. We demonstrate that the expression of UspA2/2V with the CEACAM-binding domain enable Mx to bind both to cell surface CEACAMs and to integrins, the latter via vitronectin. Such properties of UspA2/2V have not been reported to date. The studies demonstrate that the UspA family is much more heterogeneous than previously believed and illustrate the in vivo potential for exchange of functional regions between UspA proteins which could convey novel adhesive functions whilst enhancing immune evasion.

Meningococcal ligands and molecular targets of the host.

Meningococcal mechanisms of adhesion are complex, involving multiple adhesins and their respective target receptors on host cells. Three major surface structures--pili, Opa, and Opc--have been known for some time to mediate meningococcal adhesion to target human cells. More recently, several other relatively minor adhesins have also come to light. The literature on bacterial adhesion mechanisms provides numerous examples of various adhesins acting cooperatively in an apparently hierarchical and sequential manner; in other instances, adhesins may act in concert leading to high avidity interactions, often a prelude to cellular invasion and tissue penetration. Such examples are also present in the case of meningococci, although our knowledge of adhesin cooperation and synergy is far from complete. Meningococcal mechanisms used to target the host, which are often specific for the host or a tissue within the host, include both lectin-like interactions and protein-protein interactions; the latter tend to determine specificity in general. Understanding (a) what determines specificity (i.e. molecular features of adhesins and receptors), (b) encourages cellular penetration (i.e. adhesin pairs, which act in concert or synergistically to deliver effective signals for invasion and induce other cellular responses), (c) level of redundancy (more than one mechanisms of targeting host receptors), (d) host situations that encourage tissue penetration (inflammatory situations during which circulating cytokines upregulate target cell receptors, effectively encouraging greater adhesion/invasion), and (e) down-stream effects on host functions in general are all clearly important in our future strategies of controlling meningococcal pathogenesis.

Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease.

The human species is the only natural host of Neisseria meningitidis, an important cause of bacterial meningitis globally, and, despite its association with devastating diseases, N. meningitidis is a commensal organism found frequently in the respiratory tract of healthy individuals. To date, antibiotic resistance is relatively uncommon in N. meningitidis isolates but, due to the rapid onset of disease in susceptible hosts, the mortality rate remains approx. 10%. Additionally, patients who survive meningococcal disease often endure numerous debilitating sequelae. N. meningitidis strains are classified primarily into serogroups based on the type of polysaccharide capsule expressed. In total, 13 serogroups have been described; however, the majority of disease is caused by strains belonging to one of only five serogroups. Although vaccines have been developed against some of these, a universal meningococcal vaccine remains a challenge due to successful immune evasion strategies of the organism, including mimicry of host structures as well as frequent antigenic variation. N. meningitidis express a range of virulence factors including capsular polysaccharide, lipopolysaccharide and a number of surface-expressed adhesive proteins. Variation of these surface structures is necessary for meningococci to evade killing by host defence mechanisms. Nonetheless, adhesion to host cells and tissues needs to be maintained to enable colonization and ensure bacterial survival in the niche. The aims of the present review are to provide a brief outline of meningococcal carriage, disease and burden to society. With this background, we discuss several bacterial strategies that may enable its survival in the human respiratory tract during colonization and in the blood during infection. We also examine several known meningococcal adhesion mechanisms and conclude with a section on the potential processes that may operate in vivo as meningococci progress from the respiratory niche through the blood to reach the central nervous system.

Meningococcal interactions with the host.

Neisseria meningitidis interacts with host tissues through hierarchical, concerted and co-ordinated actions of a number of adhesins; many of which undergo antigenic and phase variation, a strategy that helps immune evasion. Three major structures, pili, Opa and Opc predominantly influence bacterial adhesion to host cells. Pili and Opa proteins also determine host and tissue specificity while Opa and Opc facilitate efficient cellular invasion. Recent studies have also implied a role of certain adhesin-receptor pairs in determining increased host susceptibility to infection. This chapter examines our current knowledge of meningococcal adhesion and invasion mechanisms particularly related to human epithelial and endothelial cells which are of primary importance in the disease process.

The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil.

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection.