Enterococcal bacteriophage: a survey of the tail associated lysin landscape

Bacteriophages are viruses that exclusively infect bacteria which require local degradation of cell barriers. This degradation is accomplished by various lysins located mainly within the phage tail structure. In this paper we surveyed and analysed the genomes of 506 isolated bacteriophage and prophage infecting or harboured within the genomes of the medically important Enterococcus faecalis and faecium. We highlight and characterise the major features of the genomes of phage in the morphological groups podovirus, siphovirus and myovirus, and explore their categorisation according to the new ICTV classifications, with a focus on putative extracellular lysins chiefly within tail modules. Our analysis reveals a range of potential cell-wall targeting enzyme domains that are part of tail, tape measure or other predicted base structures of these phages or prophages. These largely fall into protein domains targeting pentapeptide or glycosidic linkages within peptidoglycan but also potentially the enterococcal polysaccharide antigen (EPA) and wall teichoic acids of these species (i.e., Pectinesterases and Phosphodiesterases). Notably, there is a great variety of domain architectures that reveal the diversity of evolutionary solutions to attack the Enterococcus cell wall. Despite this variety, most phage and prophage possess a putative endopeptidase (70%), reflecting the ubiquity of this cell surface barrier. We also identified a predicted lytic transglycosylase domain belonging to the glycosyl hydrolase (GH) family 23 and present exclusively within tape measure proteins. Our data also reveal distinct features of the genomes of podo-, sipho- and myo-type viruses that most likely relate to their size and complexity. Overall, we lay a foundation for expression of recombinant TAL proteins and engineering of enterococcal and other phage that will be invaluable for researchers in this field

Sialic acid transporter NanT participates in Tannerella forsythia biofilm formation and survival on epithelial cells

Tannerella forsythia is a periodontal pathogen implicated in periodontitis. This gram-negative pathogen depends on exogenous peptidoglycan amino sugar N-acetylmuramic acid (NAM) for growth. In the biofilm state the bacterium can utilize sialic acid (Neu5Ac) instead of NAM to sustain its growth. Thus, the sialic acid utilization system of the bacterium plays a critical role in the growth and survival of the organism in the absence of NAM. We sought the function of a T. forsythia gene annotated as nanT coding for an inner-membrane sugar transporter located on a sialic acid utilization genetic cluster. To determine the function of this putative sialic acid transporter, an isogenic nanT-deletion mutant generated by allelic replacement strategy was evaluated for biofilm formation on NAM or Neu5Ac, and survival on KB epithelial cells. Moreover, since T. forsythia forms synergistic biofilms with Fusobacterium nucleatum, co-biofilm formation activity in mixed culture and sialic acid uptake in culture were also assessed. The data showed that the nanT-inactivated mutant of T. forsythia was attenuated in its ability to uptake sialic acid. The mutant formed weaker biofilms compared to the wild-type strain in the presence of sialic acid and as co-biofilms with F. nucleatum. Moreover, compared to the wild-type T. forsythia nanT-inactivated mutant showed reduced survival when incubated on KB epithelial cells. Taken together, the data presented here demonstrate that NanT-mediated sialic transportation is essential for sialic acid utilization during biofilm growth and survival of the organism on epithelial cells and implies sialic acid might be key for its survival both in subgingival biofilms and during infection of human epithelial cells in vivo

Identification of a novel N-acetylmuramic acid (MurNAc) transporter in Tannerella forsythia.

Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) that form the disaccharide-repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia. coli MurQ etherase involved in the conversion of MurNAc-6P to GlcNAc-6P. The genes encoding these components were identified on a three gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans, but not individually rescued the inability of an E. coli mutant deficient in the PTS (phosphotransferase system)-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE: In this study we report the identification of a novel transporter for peptidoglycan amino-sugar N-acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular require a PTS phosho-relay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far unique to T. forsythia, may be present in a range of bacteria both of the oral cavity and gut especially of the phylum Bacteroidetes

Identification of a Czc-like operon of the periodontal pathobiont Porphyromonas gingivalis involved in metal ion efflux

Objective The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. Methods The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function. Results Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A ΔPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm formation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2+ (500 μM), Co2+ (2 mM), and Cd2+(0.1 mM) but not Cu2+. Conclusions We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans

Porphyromonas gingivalis outer membrane vesicles increase vascular permeability

Periodontitis is increasingly associated with increased risk of cardiovascular and other systemic diseases. The Gram-negative anaerobe, Porphyromonas gingivalis, is a key periodontal pathogen, and several lines of evidence link the presence of this bacterium in the circulation with vascular disease. The outer membrane vesicles (OMVs) produced by P. gingivalis have been shown to play a role in periodontitis, although, to date, little is known about their interaction with the vasculature; therefore, this study assessed the effects of P. gingivalis OMVs on the endothelium. OMVs were isolated from wild-type strain W83 and the gingipain-deficient strain ΔK/R-ab. Immunoblotting along with cryo-EM showed gingipain expression in W83 but not ΔK/R-ab-derived OMVs, where gingipains were localized to the cell wall surface. Confluent endothelial cell monolayers infected with either W83 or W83-derived OMV displayed significantly increased dextran permeability over those infected with ΔK/R-ab or its OMV. Moreover, W83-derived OMVs induced significantly more vascular disease in a zebrafish larvae systemic infection model over 72 h compared to those injected with gingipain-deficient OMVs or controls. In line with these data, human microvascular endothelial cells (HMEC-1) displayed an OMV-associated, gingipain-dependent decrease in cell surface levels of the intercellular adhesion molecule PECAM-1 (CD31) when examined by flow cytometry. These data show, for the first time, that OMVs from P. gingivalis mediate increased vascular permeability, leading to a diseased phenotype both in vitro and in vivo. Moreover, these data strongly implicate gingipains present on the OMV surface in mediating these vascular events, most likely via a mechanism that involves proteolytic cleavage of endothelial cell-cell adhesins such as PECAM-1. These data provide important evidence for the role of bacterial-derived OMVs in mediating systemic disease

Protein-linked glycans in periodontal bacteria : prevalence and role at the immune interface

Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendritic cell cytokine responses to drive T cell immunity in ways that facilitate bacterial persistence in the host and induce periodontal inflammation. In addition, surface glycans may help certain periodontal bacteria protect against serum complement attack or help them escape immune detection through glycomimicry. In this review we will focus mainly on the generalized surface-layer protein glycosylation system of the periodontal pathogen Tannerella forsythia in shaping innate and adaptive host immunity in the context of periodontal disease. In addition, we will also review the current state of knowledge of surface protein glycosylation and its potential for immune modulation in other periodontal pathogens

Draft Genome Sequences of Three Clinical Isolates of Tannerella forsythia Isolated from Subgingival Plaque from Periodontitis Patients in the United States.

We report the genome sequences of three clinical isolates of Tannerella forsythia from the subgingival plaque of periodontitis patients attending clinics at the School of Dental Medicine, University at Buffalo. The availability of these genome sequences will aid the understanding of the pathogenesis of periodontitis

Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia

Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis

The dental plaque biofilm matrix

The extracellular matrix is a critical component of microbial biofilms, such as dental plaque, maintaining the spatial arrangement of cells and coordinating cellular functions throughout the structure. The extracellular polymeric substances that comprise the matrix include carbohydrates, nucleic acids, proteins, and lipids, which are frequently organized into macromolecular complexes and/or are associated with the surfaces of microbial cells within the biofilm. Cariogenic dental plaque is rich in glucan and fructan polysaccharides derived from extracellular microbial metabolism of dietary sucrose. By contrast, the matrix of subgingival dental plaque is a complex mixture of macromolecules that is still not well understood. Components of the matrix escape from microbial cells during lysis by active secretion or through the shedding of vesicles and serve to anchor microbial cells to the tooth surface. By maintaining the biofilm in close association with host tissues, the matrix facilitates interactions between microorganisms and the host. The outcome of these interactions may be the maintenance of health or the development of dental disease, such as caries or periodontitis. The matrix affords microbial cells protection against chemical and physical insults and hinders the eradication of pathogenic dental plaque. Therefore, strategies to control the matrix are critical to maintain oral health. This review discusses recent advances in our understanding of the composition, origins, and function of the dental plaque matrix, with a focus on subgingival dental plaque. New strategies to control subgingival dental plaque based on targeting the biofilm matrix are also considered

Characterization of an alpha-L-fucosidase from the periodontal pathogen Tannerella forsythia

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 μM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase