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.

Bi-directional signaling between the intestinal epithelium and type-3 innate lymphoid cells regulates secretory dynamics and interleukin-22.

Type-3 innate lymphoid cells (ILC3) respond to localized environmental cues to regulate homeostasis and orchestrate immunity in the intestine. The intestinal epithelium is an important upstream regulator and downstream target of ILC3 signaling, however, the complexity of mucosal tissues can hinder efforts to define specific interactions between these two compartments. Here, we employ a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3 to uncover bi-directional signaling mechanisms that underlie intestinal homeostasis. We report that ILC3 induce global transcriptional changes in intestinal epithelial cells, driving the enrichment of secretory goblet cell signatures. We find that SIO enriched for goblet cells promote NKp46+ ILC3 and interleukin (IL)-22 expression, which can feedback to induce IL-22-mediated epithelial transcriptional signatures. However, we show that epithelial regulation of ILC3 in this system is contact-dependent and demonstrate a role for epithelial Delta-Like-Canonical-Notch-Ligand (Dll) in driving IL-22 production by ILC3, via subset-specific Notch1-mediated activation of T-bet+ ILC3. Finally, by interfering with Notch ligand-receptor dynamics, ILC3 appear to upregulate epithelial Atoh1 to skew secretory lineage determination in SIO-ILC3 co-cultures. This research outlines two complimentary bi-directional signaling modules between the intestinal epithelium and ILC3, which may be relevant in intestinal homeostasis and disease.

Hemin availability induces coordinated DNA methylation and gene expression changes in Porphyromonas gingivalis.

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

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.

Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.

Molecular basis for avirulence of spontaneous variants of Porphyromonas gingivalis: Genomic analysis of strains W50, BE1 and BR1.

The periodontal pathogen Porphyromonas gingivalis is genetically heterogeneous. However, the spontaneous generation of phenotypically different sub-strains has also been reported. McKee et al. (1988) cultured P. gingivalis W50 in a chemostat during investigations into the growth and properties of this bacterium. Cell viability on blood agar plates revealed two types of non-pigmenting variants, W50 beige (BE1), and W50 brown (BR1), in samples grown in a high-hemin medium after day 7, and the population of these variants increased to approximately 25% of the total counts by day 21. W50, BE1 and BR1 had phenotypic alterations in pigmentation, reduced protease activity and haemagglutination and susceptibility to complement killing. Furthermore, the variants exhibited significant attenuation in a mouse model of virulence. Other investigators showed that in BE1, the predominant extracellular Arg-gingipain was RgpB, and no reaction with an A-lipopolysaccharide-specific MAb 1B5 (Collinson et al., 1998; Slaney et al., 2006). In order to determine the genetic basis for these phenotypic properties, we performed hybrid DNA sequence long reads using Oxford Nanopore and the short paired-end DNA sequence reads of Illumina HiSeq platforms to generate closed circular genomes of the parent and variants. Comparative analysis indicated loss of intact kgp in the 20 kb region of the hagA-kgp locus in the two variants BE1 and BR1. Deletions in hagA led to smaller open reading frames in the variants, and BR1 had incurred a major chromosomal DNA inversion. Additional minor changes to the genomes of both variants were also observed. Given the importance of Kgp and HagA to protease activity and haemagglutination, respectively, in this bacterium, genomic changes at this locus may account for most of the phenotypic alterations of the variants. The homologous and repetitive nature of hagA and kgp and the features at the inverted junctions are indicative of specific and stable homologous recombination events, which may underlie the genetic heterogeneity of this species.

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.

The role of oral bacteria in inflammatory bowel disease.

Over the past two decades, the importance of the microbiota in health and disease has become evident. Pathological changes to the oral bacterial microbiota, such as those occurring during periodontal disease, are associated with multiple inflammatory conditions, including inflammatory bowel disease. However, the degree to which this association is a consequence of elevated oral inflammation or because oral bacteria can directly drive inflammation at distal sites remains under debate. In this Perspective, we propose that in inflammatory bowel disease, oral disease-associated bacteria translocate to the intestine and directly exacerbate disease. We propose a multistage model that involves pathological changes to the microbial and immune compartments of both the oral cavity and intestine. The evidence to support this hypothesis is critically evaluated and the relevance to other diseases in which oral bacteria have been implicated (including colorectal cancer and liver disease) are discussed.

Quantification of Workload and Wellness Measures in a Women's Collegiate Volleyball Season.

The purpose of this paper was to quantify internal and external loads completed by collegiate volleyball athletes during a competitive season. Eleven players were sampled (using accelerometers and subjective wellness surveys) during the practice (n = 55) and game (n = 30) sessions over the 2019 season. Longitudinal data were evaluated for trends across the preseason, non-conference play, and conference play periods. Data were also analyzed with respect to positional groups. Longitudinal analysis of accelerometer data showed higher workload demand during practices than games. Positional group differences were most when evaluating jump count and height. Setters accrued over twice as many jumps in a practice than during a game and had similar overall jump counts in practice to attacking positions. Average team wellness values varied with time in the season, especially during times of congested travel. This is the first study to look at both game and practice workload and wellness measures in collegiate women's volleyball. The results suggest athlete monitoring can be used to understand the demands of volleyball and used in the future to enhance practice and recovery day design to optimize athlete well-being.

The Relationship between Mucins and Ulcerative Colitis: A Systematic Review.

Mucins are a family of glycosylated proteins which are the primary constituents of mucus and play a dynamic role in the regulation of the protective mucosal barriers throughout the human body. Ulcerative colitis (UC) is an Inflammatory Bowel Disease (IBD) characterised by continuous inflammation of the inner layer of the large intestine, and in this systematic review we analyse currently available data to determine whether alterations exist in mucin activity in the colonic mucosa of UC patients. Database searches were conducted to identify studies published between 1990 and 2020 that assess the role of mucins in cohorts of UC patients, where biopsy specimens were resected for analysis and control groups were included for comparison. 5497 articles were initially identified and of these 14 studies were systematically selected for analysis, a further 2 articles were identified through citation chaining. Therefore, 16 studies were critically reviewed. 13 of these studies assessed the role of MUC2 in UC and the majority of articles indicated that alterations in MUC2 structure or synthesis had an impact on the colonic mucosa, although conflicting results were presented regarding MUC2 expression. This review highlights the importance of further research to enhance our understanding of mucin regulation in UC and summarises data that may inform future studies.

Microbial transitions from health to disease.

Recent advances in our understanding of the microbial populations that colonize the human mouth, their acquisition, interdependency, and coevolution with the host, bring a different perspective to the mechanisms underpinning the maintenance of periodontal health and the development of disease. In this work we suggest that our knowledge map of the etiology of periodontal health and disease can be viewed as a broad, highly connected, and integrated system that spans the entire spectrum of microbe/host/clinical interactions. The overall concept of present Periodontology 2000, that the microbial biofilm can be considered a human tissue of bacteriological origin, is entirely consistent with this integrated system view. The health-associated community structure of microbial biofilms can be considered a system that is normally resilient to perturbation. Equally, there is evidence to suggest that the dysbiotic community structure in disease may share similar resilience properties. In both instances, the resilience may be governed by the precise makeup of the acquired microbiome and by the genetics of the host. Understanding the mechanisms that enable the resistance to change of healthy and dysbiotic microbial populations may be important in the development of approaches to prevent the progression of disease and to restore health in diseased individuals.

Oral biofilms revisited: A novel host tissue of bacteriological origin.

The central theme of this volume of Periodontology 2000 is that the microbial dental plaque biofilm, specifically the subgingival dental plaque biofilm, mimics a human tissue in both structure and function. As a basis for this assertion we use the definition of a tissue as an aggregate of similar cells and cell products forming a defined structure with a specific function, in a multicellular organism. Accordingly, we propose that the dental plaque biofilm represents an acquired human tissue largely of bacterial origin that maintains the health of gingival tissue. Furthermore, we acknowledge that disease can be defined as a deviation from the normal structure or an interruption to the function of any body part, organ, or system, and that is manifested by a characteristic set of symptoms and signs whose etiology, pathology, and prognosis may be known or unknown. Therefore, in this volume we present the concept that periodontitis is a disruption of the normal function of the healthy subgingival plaque biofilm with concomitant disruption to its functional properties in relation to innate defense surveillance and tissue maintenance, leading to excessive, deregulated inflammation and tissue destruction.

PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two ß-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the "attachment complex" composed of PorU, PorZ, and the integral OM ß-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

A 16S rRNA Gene and Draft Genome Database for the Murine Oral Bacterial Community.

A curated murine oral microbiome database to be used as a reference for mouse-based studies has been constructed using a combination of bacterial culture, 16S rRNA gene amplicon, and whole-genome sequencing. The database comprises a collection of nearly full-length 16S rRNA gene sequences from cultured isolates and draft genomes from representative taxa collected from a range of sources, including specific-pathogen-free laboratory mice, wild Mus musculus domesticus mice, and formerly wild wood mouse Apodemus sylvaticus At present, it comprises 103 mouse oral taxa (MOT) spanning four phyla-Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes-including 12 novel undescribed species-level taxa. The key observations from this study are (i) the low diversity and predominantly culturable nature of the laboratory mouse oral microbiome and (ii) the identification of three major murine-specific oral bacterial lineages, namely, Streptococcus danieliae (MOT10), Lactobacillus murinus (MOT93), and Gemella species 2 (MOT43), which is one of the novel, still-unnamed taxa. Of these, S. danieliae is of particular interest, since it is a major component of the oral microbiome from all strains of healthy and periodontally diseased laboratory mice, as well as being present in wild mice. It is expected that this well-characterized database should be a useful resource for in vitro experimentation and mouse model studies in the field of oral microbiology.IMPORTANCE Mouse model studies are frequently used in oral microbiome research, particularly to investigate diseases such as periodontitis and caries, as well as other related systemic diseases. We have reported here the details of the development of a curated reference database to characterize the oral microbial community in laboratory and some wild mice. The genomic information and findings reported here can help improve the outcomes and accuracy of host-microbe experimental studies that use murine models to understand health and disease. Work is also under way to make the reference data sets publicly available on a web server to enable easy access and downloading for researchers across the world.

The mouth opens wide onto an additional view of immune development at mucosal borders.

Much of our understanding of the way mucosal surfaces achieve a harmonious balance with their resident commensal microbiota derives from analysis of this interplay in the gut. Koren et al. (2021) interrogate the dynamics of this relationship in the mouth during early life and find that highly tissue-specific responses facilitate maturation.

Inflammatory arthritis disrupts gut resolution mechanisms, promoting barrier breakdown by Porphyromonas gingivalis.

Rheumatoid arthritis is linked with altered host immune responses and severe joint destruction. Recent evidence suggests that loss of gut homeostasis and barrier breach by pathobionts, including Porphyromonas gingivalis, may influence disease severity. The mechanism(s) leading to altered gut homeostasis and barrier breakdown in inflammatory arthritis are poorly understood. In the present study, we found a significant reduction in intestinal concentrations of several proresolving mediators during inflammatory arthritis, including downregulation of the gut-protective mediator resolvin D5n-3 DPA (RvD5n-3 DPA). This was linked with increased metabolism of RvD5n-3 DPA to its inactive 17-oxo metabolite. We also found downregulation of IL-10 expression in the gut of arthritic mice that was coupled with a reduction in IL-10 and IL-10 receptor (IL-10R) in lamina propria macrophages. These changes were linked with a decrease in the number of mucus-producing goblet cells and tight junction molecule expression in the intestinal epithelium of arthritic mice when compared with naive mice. P. gingivalis inoculation further downregulated intestinal RvD5n-3 DPA and Il-10 levels and the expression of gut tight junction proteins. RvD5n-3 DPA, but not its metabolite 17-oxo-RvD5n-3 DPA, increased the expression of both IL-10 and IL-10R in macrophages via the upregulation of the aryl hydrocarbon receptor agonist l-kynurenine. Administration of RvD5n-3 DPA to arthritic P. gingivalis-inoculated mice increased intestinal Il-10 expression, restored gut barrier function, and reduced joint inflammation. Together, these findings uncover mechanisms in the pathogenesis of rheumatoid arthritis, where disruption of the gut RvD5n-3 DPA-IL-10 axis weakens the gut barrier, which becomes permissive to the pathogenic actions of the pathobiont P. gingivalis.

Leadership in preparations for the Research Excellence Framework.

The Research Excellence Framework (REF) 2021 will inform the allocation of public funds for research to UK dental schools until at least 2027. Although the outcome has the potential to provide a national picture of research activity and strengths in dentistry, this opportunity may be lost given changes in the criteria for REF submission. Preparations for a successful REF are high on the agenda of all schools. This article presents some of the leadership challenges posed by REF2021.

Noninvasive Assessment of Internal and External Player Load: Implications for Optimizing Athletic Performance.

Heishman, AD, Curtis, MA, Saliba, E, Hornett, RJ, Malin, SK, and Weltman, AL. Noninvasive assessment of internal and external player load: implications for optimizing athletic performance. J Strength Cond Res 32(5): 1280-1287, 2018-Few data exist that assess athlete tracking and monitoring for the development of strategies to optimize performance and reduce fatigue in elite athletes. The purpose of the present study was to assess the efficacy of external load and internal stress monitoring as assessment tools for examining a performance index of fatigue. A retrospective analysis was performed on data collected over the course of the preseason in 10 elite male NCAA Division 1 basketball players. Internal stress was assessed using Omegawave Technology readiness scores and compared with the performance index of countermovement jump (CMJ). The external load that accumulated during the previous practice, quantified by PlayerLoad (PL; Catapult), was compared with CMJ values and Omegawave scores. The results indicated that high, compared to low CNS Omegawave Readiness Scores (6.7 ± 05.1, 4.5 ± 1.2 AU; p < 0.001), were associated with increased CMJ (62.1 ± 6.5 vs. 59.4 ± 6.6 cm; p = 0.05), Power (6,590 ± 526.7 vs. 6,383.5 ± 606.8 W; p = 0.05), Omegawave Overall Readiness (5.8 ± 1.1 vs. 5.0 ± 0.7 AU; p = 0.05), and Omega Potential (Omega) (21.3 ± 6.3 vs. 9.9 ± 20.8 mV; p = 0.07). An increased PL during the previous exposure was associated with decreased CMJ (58.7 ± 4.7 cm vs. 60.4 ± 5.1 cm; p < 0.001) and increased TRIMP (135.1 ± 35.9 vs. 65.6 ± 20.0 AU; p < 0.001), and duration (115.4 ± 27.1 vs. 65.56 ± 20.0 minutes; p = < 0.001) despite no differences in Omegawave CNS Readiness scores. We conclude that Omegawave and Catapult technologies provide independent information related to performance and may be effective tools for monitoring athlete performance.