Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

Comparative Microbial Profiles of Caries and Black Extrinsic Tooth Stain in Primary Dentition.

Extrinsic black tooth stain (BS) is a common oral disease associated with lower caries experience in preschool children, although the microbiotic features contributing to the low risk of caries in this group remain elusive. In this study, we aimed at identifying the dominant bacteria in dental plaque to indicate the incidence of caries in the primary dentition. Subjects were divided into 3 groups based on the clinical examination: group CF, children without pigment who had no caries lesions or restorations (n = 18); group CS, children who were diagnosed with severe early childhood caries (n = 17); and group BS, children with pigment (black extrinsic stain) without caries or restorations (n = 15). The total microbial genomic DNA was extracted and subjected to bacterial 16S ribosomal RNA gene sequencing using an Illumina HiSeq platform. The differential dominant bacteria were determined using Wilcoxon rank-sum testing and linear discriminant analysis effect size (LEfSe). Co-occurrence network analysis was performed using sparse correlations for compositional data, calculation and functional features were predicted using PICRUSt. Interestingly, our results showed that the relative abundance of Pseudopropionibacterium, Actinomyces, Rothia, and Cardiobacterium was from high to low and that of Porphyromonas was low to high in the BS, CF, and CS groups, consistent with the clinical incidence of caries in the 3 groups. Moreover, an increased level of Selenomonas_3, Fusobacterium, and Leptotrichia was associated with high caries prevalence. We found that the interactions among genera in the BS and CS plaque communities are less complex than those in the CF communities at the taxon level. Functional features, including cofactor and vitamin metabolism, glycan biosynthesis and metabolism, and translation, significantly increased in caries plaque samples. These bacterial competition- and commensalism-induced changes in microbiota would result in a change of their symbiotic function, finally affecting the balance of oral microflora.

Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals.

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.

Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis.

Gram-negative pathogens express fibrous adhesive organelles that mediate targeting to sites of infection. The major class of these organelles is assembled via the classical, alternative and archaic chaperone-usher pathways. Although non-classical systems share a wider phylogenetic distribution and are associated with a range of diseases, little is known about their assembly mechanisms. Here we report atomic-resolution insight into the structure and biogenesis of Acinetobacter baumannii Csu and Escherichia coli ECP biofilm-mediating pili. We show that the two non-classical systems are structurally related, but their assembly mechanism is strikingly different from the classical assembly pathway. Non-classical chaperones, unlike their classical counterparts, maintain subunits in a substantially disordered conformational state, akin to a molten globule. This is achieved by a unique binding mechanism involving the register-shifted donor strand complementation and a different subunit carboxylate anchor. The subunit lacks the classical pre-folded initiation site for donor strand exchange, suggesting that recognition of its exposed hydrophobic core starts the assembly process and provides fresh inspiration for the design of inhibitors targeting chaperone-usher systems.

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system.

Many Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type II secretion.

Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes.

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1-SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.

Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.

The peptide-binding cavity is essential for Als3-mediated adhesion of Candida albicans to human cells.

The adhesive phenotype of Candida albicans contributes to its ability to colonize the host and cause disease. Als proteins are one of the most widely studied C. albicans virulence attributes; deletion of ALS3 produces the greatest reduction in adhesive function. Although adhesive activity is thought to reside within the N-terminal domain of Als proteins (NT-Als), the molecular mechanism of adhesion remains unclear. We designed mutations in NT-Als3 that test the contribution of the peptide-binding cavity (PBC) to C. albicans adhesion and assessed the adhesive properties of other NT-Als3 features in the absence of a functional PBC. Structural analysis of purified loss-of-PBC-function mutant proteins showed that the mutations did not alter the overall structure or surface properties of NT-Als3. The mutations were incorporated into full-length ALS3 and integrated into the ALS3 locus of a deletion mutant, under control of the native ALS3 promoter. The PBC mutant phenotype was evaluated in assays using monolayers of human pharyngeal epithelial and umbilical vein endothelial cells, and freshly collected human buccal epithelial cells in suspension. Loss of PBC function resulted in an adhesion phenotype that was indistinguishable from the Δals3/Δals3 strain. The adhesive contribution of the Als3 amyloid-forming-region (AFR) was also tested using these methods. C. albicans strains producing cell surface Als3 in which the amyloidogenic potential was destroyed showed little contribution of the AFR to adhesion, instead suggesting an aggregative function for the AFR. Collectively, these results demonstrate the essential and principal role of the PBC in Als3 adhesion.

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus.

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

The Legionella collagen-like protein employs a distinct binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual trimer arrangement with a positively charged external surface and negatively charged solvent exposed internal cavity. Through molecular dynamics simulations, we show how the glycosaminoglycan chondroitin-4-sulphate associates with the Lcl-CTD surface via distinct binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate-binding mechanism.

I shall be released: Assembly platform dynamics during type II secretion.

In this issue of Structure, Dazzoni et al. solve the high-resolution homo- and hetero-dimeric structures of the Klebsiella oxytoca PulL and PulM C-terminal domains and unravel an uncharacterized dynamic interaction interface that is required for correct function of the type II secretion system.

How to study biofilms: technological advancements in clinical biofilm research.

Biofilm formation is an important survival strategy commonly used by bacteria and fungi, which are embedded in a protective extracellular matrix of organic polymers. They are ubiquitous in nature, including humans and other animals, and they can be surface- and non-surface-associated, making them capable of growing in and on many different parts of the body. Biofilms are also complex, forming polymicrobial communities that are difficult to eradicate due to their unique growth dynamics, and clinical infections associated with biofilms are a huge burden in the healthcare setting, as they are often difficult to diagnose and to treat. Our understanding of biofilm formation and development is a fast-paced and important research focus. This review aims to describe the advancements in clinical biofilm research, including both in vitro and in vivo biofilm models, imaging techniques and techniques to analyse the biological functions of the biofilm.

The influence of ions on the lubricative abilities of mucin and the role of sialic acids.

Mucus reduces friction between epithelial surfaces by providing lubrication in the boundary and mixed regime. Mucins, the main macromolecule, are heavily glycosylated proteins that polymerise and retain water molecules, resulting in a hydrated biogel. It is assumed that positively charged ions can influence mucin film structure by screening the electrostatic repulsions between the negatively charged glycans on mucin moieties and draw in water molecules via hydration shells. The ionic concentration can vary significantly in different mucus systems and here we show that increasing the ionic concentration in mucin films leads to an increase in lubrication between two polydimethylsiloxane surfaces at sliding contact in a compliant oral mimic. Mucins were found to bind sodium ions in a concentration-dependent manner and increased ionic concentration appears to cause mucin films to swell when assessed by Quartz Crystal hiMicrobalance with Dissipation (QCM-D) analysis. Furthermore, we determined that the removal of negatively charged sialic acid moieties by sialidase digestion resulted in reduced adsorption to hydrophilic surfaces but did not affect the swelling of mucin films with increasing ionic concentrations. Moreover, the coefficient of friction was increased with sialic acid removal, but lubrication was still increased with increasing ionic concentrations. Taken together this suggests that sialic acids are important for lubrication and may exert this through the sacrificial layer mechanism. Ionic concentration appears to influence mucin films and their lubrication, and sialic acids, at least partly, may be important for ion binding.

The Legionella collagen-like protein employs a unique binding mechanism for the recognition of host glycosaminoglycans.

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires' disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans (GAGs) on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual dynamic trimer arrangement with a positively charged external surface and a negatively charged solvent exposed internal cavity. Through Molecular Dynamics (MD) simulations, we show how the GAG chondroitin-4-sulphate associates with the Lcl-CTD surface via unique binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate binding mechanism.

Structural insight into the role of Streptococcus parasanguinis Fap1 within oral biofilm formation.

The fimbriae-associated protein 1 (Fap1) is a major adhesin of Streptococcus parasanguinis, a primary colonizer of the oral cavity that plays an important role in the formation of dental plaque. Fap1 is an extracellular adhesive surface fibre belonging to the serine-rich repeat protein (SRRP) family, which plays a central role in the pathogenesis of streptococci and staphylococci. The N-terminal adhesive region of Fap1 (Fap1-NR) is composed of two domains (Fap1-NR(α) and Fap1-NR(ß)) and is projected away from the bacterial surface via the extensive serine-rich repeat region, for adhesion to the salivary pellicle. The adhesive properties of Fap1 are modulated through a pH switch in which a reduction in pH results in a rearrangement between the Fap1-NR(α) and Fap1-NR(ß) domains, which assists in the survival of S. parasanguinis in acidic environments. We have solved the structure of Fap1-NR(α) at pH 5.0 at 3.0Ǻ resolution and reveal how subtle rearrangements of the 3-helix bundle combined with a change in electrostatic potential mediates 'opening' and activation of the adhesive region. Further, we show that pH-dependent changes are critical for biofilm formation and present an atomic model for the inter-Fap1-NR interactions which have been assigned an important role in the biofilm formation.

Promoting crystallisation of the Salmonella enteritidis fimbriae 14 pilin SefD using deuterium oxide.

The use of heavy water (D(2)O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D(2)O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex.

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane ß-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation.

Escherichia coli is a Gram-negative bacterium that colonises the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here, we examine SslE from the commensal E. coli Waksman and BL21 (DE3) strains and the enterotoxigenic H10407 and enteropathogenic E2348/69 strains. We reveal that SslE has a unique and dynamic structure in solution and in response to acidification within mature biofilms it can form a unique aggregate with amyloid-like properties. Furthermore, we show that both SslE monomers and aggregates bind DNA in vitro and co-localise with extracellular DNA (eDNA) in mature biofilms, and SslE aggregates may also associate with cellulose under certain conditions. Our results suggest that interactions between SslE and eDNA are important for biofilm maturation in many E. coli strains and SslE may also be a factor that drives biofilm formation in other SslE-secreting bacteria.