A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

Major pathogenic clonal complexes (cc) of Neisseria meningitidis differ substantially in their point prevalence among healthy carriers. We show that frequently carried pathogenic cc (e.g. sequence type ST‐41/44 cc and ST‐32 cc) depend on extracellular DNA (eDNA) to initiate in vitro biofilm formation, whereas biofilm formation of cc with low point prevalence (ST‐8 cc and ST‐11 cc) was eDNA‐independent. For initial biofilm formation, a ST‐32 cc type strain, but not a ST‐11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N‐acetylmuramyl‐l ‐alanine amidase genes. In late biofilms, outer membrane phospholipase A‐dependent autolysis, which was observed in most cc, but not in ST‐8 and ST‐11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an eDNA‐dependent one yielding shear force resistant microcolonies, and an eDNA‐independent one. Based on the experimental findings and previous epidemiological observations, we hypothesize that most meningococcal cc display a settler phenotype, which is eDNA‐dependent and results in a stable interaction with the host. On the contrary, spreaders (ST‐11 and ST‐8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates.     

Evaluation of the kinetics and mechanism of action of anti‐integration host factor‐mediated disruption of bacterial biofilms

The extracellular polymeric substance produced by many human pathogens during biofilm formation often contains extracellular DNA (eDNA). Strands of bacterial eDNA within the biofilm matrix can occur in a lattice‐like network wherein a member of the DNABII family of DNA‐binding proteins is positioned at the vertex of each crossed strand. To date, treatment of all biofilms tested with antibodies directed against one DNABII protein, Integration Host Factor (IHF), results in significant disruption. Here, using non‐typeable Haemophilus influenzae as a model organism, we report that this effect was rapid, IHF‐specific and mediated by binding of transiently dissociated IHF by anti‐IHF even when physically separated from the biofilm by a nucleopore membrane. Further, biofilm disruption fostered killing of resident bacteria by previously ineffective antibiotics. We propose the mechanism of action to be the sequestration of IHF upon dissociation from the biofilm eDNA, forcing an equilibrium shift and ultimately, collapse of the biofilm. Further, antibodies against a peptide positioned at the DNA‐binding tips of IHF were as effective as antibodies directed against the native protein. As incorporating eDNA and associated DNABII proteins is a common strategy for biofilms formed by multiple human pathogens, this novel therapeutic approach is likely to have broad utility.     

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm‐negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and eDNA‐dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.     

PrgB promotes aggregation,biofilm formation,and conjugation through DNA binding and compaction

Gram‐positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram‐positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono‐ or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell‐cell contacts through alternative binding of cell‐bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra‐ or inter‐species T4SS‐mediated gene transfer.     

Enterococcus faecalis pCF10‐encoded surface proteins PrgA,PrgB (aggregation substance) and PrgC contribute to plasmid transfer,biofilm formation and virulence

Enterococcus faecalis pCF10 transfers at high frequencies upon pheromone induction of the prgQ transfer operon. This operon codes for three cell wall‐anchored proteins – PrgA, PrgB (aggregation substance) and PrgC – and a type IV secretion system through which the plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to plasmid transfer, biofilm formation and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 transfer at wild‐type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone‐responsive, conjugative plasmids of E. faecalis have retained Prg‐like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation and widespread lateral gene transfer.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α‐melanocyte stimulating hormone (α‐MSH) is a tridecapeptide fragment of pro‐opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti‐inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro‐inverso (RI) sequence of α‐MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI‐α‐MSH exhibited a diminished binding affinity for MC1R compared to α‐MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non‐natural amino acids and substitution of the C‐terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical K_i as α‐MSH at MC1R and a lower EC₅₀ in Mel‐624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L ‐form α‐MSH, suggesting a significantly altered interaction with the MC1R. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms

A novel benzimidazole molecule that was identified in a small‐molecule screen and is known as antibiofilm compound 1 (ABC‐1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC‐1 was tested in various biofilm‐forming strains of S. aureus. It was demonstrated that ABC‐1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall‐associated protein dependent or cell wall‐ associated extracellular DNA (eDNA). Of note, ABC‐1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC‐1 treated strains, implying that ABC‐1 affects not only SpA but also other factors. Indeed, ABC‐1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC‐1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.     

Characteristics of C‐terminal, β‐amyloid peptide binding fragment of neuroprotective protease inhibitor,cystatin C

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC‐induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C‐terminal fragment hCC(101‐117) was identified as the Aβ‐binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17‐24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme‐linked immunosorbent assay‐like assay, by surface plasmon resonance, and by nano‐ESI‐FTICR MS of the hCC–Aβ‐ binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101‐117), revealed the highest relative inhibitory effect toward Aβ‐fibril formation. Herein, we present further studies on molecular details of the hCC‐Aβ complex. With Ala substitution, affinity experiments, and enzyme‐linked immunosorbent assay‐like assays for the Aβ‐binding fragment, hCC(101‐117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101‐117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Enterococcus adhesin PrgB facilitates type IV secretion by condensation of extracellular DNA

Conjugative type IV secretion systems (T4SSs) are multi‐protein complexes in Gram‐negative and Gram‐positive (G+) bacteria, responsible for spreading antibiotic resistances and virulence factors among different species. Compared to Gram‐negative bacteria, which establish close contacts for conjugative transfer via sex pili, G+ T4SSs are suggested to employ surface adhesins instead. One example is pCF10, an enterococcal conjugative sex‐pheromone responsive plasmid with a narrow host range, thus disseminating genetic information only among closely related species. This MicroCommentary is dedicated to the crystal structure of the pCF10‐encoded adhesion domain of PrgB presented by Schmitt et al. The authors show in their work that this adhesion domain is responsible for biofilm formation, tight binding and condensation of extracellular DNA (eDNA) and conjugative transfer of pCF10. A sophisticated two‐step mechanism for highly efficient conjugative transfer is postulated, including the formation of PrgB‐mediated long‐range intercellular contacts by binding and establishment of shorter‐range contacts via condensation of eDNA. PrgB binding to lipoteichoic acid on the recipient cell surface stabilizes junctions between the mating partners. The major findings by Schmitt et al. will be brought into a broader context and potential medical applications targeting eDNA as essential component in biofilm formation and conjugation will be discussed.     

Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms

Persistent staphylococcal infections often involve surface‐associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co‐ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.     

Pathogenic factors in Candida biofilm‐related infectious diseases

Candida albicans is a commonly found member of the human microflora and is a major human opportunistic fungal pathogen. A perturbation of the microbiome can lead to infectious diseases caused by various micro‐organisms, including C. albicans. Moreover, the interactions between C. albicans and bacteria are considered to play critical roles in human health. The major biological feature of C. albicans, which impacts human health, resides in its ability to form biofilms. In particular, the extracellular matrix (ECM) of Candida biofilm plays a multifaceted role and therefore may be considered as a highly attractive target to combat biofilm‐related infectious diseases. In addition, extracellular DNA (eDNA) also plays a crucial role in Candida biofilm formation and its structural integrity and induces the morphological transition from yeast to the hyphal growth form during C. albicans biofilm development. This review focuses on pathogenic factors such as eDNA in Candida biofilm formation and its ECM production and provides meaningful information for future studies to develop a novel strategy to battle infectious diseases elicited by Candida‐formed biofilm.     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Extracellular DNA in Helicobacter pylori biofilm: a backstairs rumour

Aims: This study detected and characterized the extracellular DNA (eDNA) in the biofilm extracellular polymeric substance (EPS) matrix of Helicobacter pylori and investigated the role of such component in the biofilm development. Methods and Results: Extracellular DNA was purified and characterized in a 2‐day‐old mature biofilm developed by the reference strain H. pylori ATCC 43629, the clinical isolate H. pylori SDB60 and the environmental strain H. pylori MDC1. Subsequently, the role of eDNA in the H. pylori biofilm was evaluated by adding DNase I during biofilm formation and on mature biofilms. Extracellular DNA was detected in the 2‐day‐old EPS biofilm matrix of all analysed H. pylori strains. The DNA fingerprintings, performed by RAPD analysis, on eDNA and intracellular DNA (iDNA), showed some remarkable differences. The data obtained by microtitre biofilm assay as well as colony forming unit count and CLSM (confocal laser scanning microscopy) qualitative analysis did not show any significant differences between the DNase I‐treated biofilms and the corresponding not treated controls both in formation and on mature biofilms. Conclusions: In this study, we provide evidence that eDNA is a component of the EPS matrix of H. pylori biofilm. The different profiles of eDNA and iDNA indicate that lysed cells are not the primary source of eDNA release, suggesting that other active mechanisms might be involved in this process. Moreover, the biomass assay suggests that eDNA may not be the main component of biofilm matrix, suggesting that it could be primarily involved in other mechanisms such as recombination processes, via transformation, contributing to the wide genomic variability of this micro‐organism defined as a ‘quasi‐species’. Significance and Impact of the Study: The presence of eDNA in H. pylori biofilm can contribute to the active dynamic exchange of information aimed to reach the best condition for the bacterial survival in the host and in the environment.     

Presence of Extracellular DNA in the <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Matrix and its Contribution to Biofilms

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.     

Protective potency of recombinant meningococcal IgA1 protease and its structural derivatives upon animal invasion with meningococcal and pneumococcal infections

Immunization of mice with recombinant IgA1 protease of Neisseria meningitidis or several structural derivatives thereof protects the animals infected with a variety of deadly pathogens, including N. meningitidis serogroups A, B, and C and 3 serotypes of Streptococcus pneumonia. In sera of rabbits immunized with inactivated pneumococcal cultures, antibodies binding IgA1-protease from N. meningitidis serogroup B were detected. Thus, the cross-reactive protection against meningococcal and pneumococcal infections has been demonstrated in vivo. Presumably it indicates the presence of common epitopes in the N. meningitidis IgA1 protease and S. pneumoniae surface proteins.     

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.     

sarA‐mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant‐associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease‐dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease‐mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.