Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage.

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM ß-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

IL-36γ regulates mediators of tissue homeostasis in epithelial cells.

IL-36 cytokines are critical regulators of mucosal inflammation and homeostasis. IL-36γ regulates the expression of inflammatory cytokines and antimicrobial proteins by gingival epithelial cells (e.g. TIGK cells). Here, we show that IL-36γ also regulates the expression of matrix metalloproteinase 9 (MMP9) and neutrophil gelatinase-associated lipocalin (NGAL), important mediators of antimicrobial immunity and tissue homeostasis in mucosal epithelia. MMP9 and NGAL were not similarly induced by IL-17 or IL-22, thus indicating the importance of IL-36γ in the regulation of MMP9 and NGAL. Mechanistically, MMP9 and NGAL expression was demonstrated to be induced in an IRAK1- and NF-κB-dependent manner. Furthermore, signaling by p38 MAP kinase may enable their expression to be independently regulated by IL-36γ. The stronger IL-36γ-inducible expression of MMP9 and NGAL in terminally differentiating TIGK cells suggests that control of their expression is associated with the maturation of the gingival epithelium. Although MMP9 and NGAL expression in epithelial cells can also be induced by bacteria, their expression in TIGK cells was not induced by the periodontal pathogen Porphyromonas gingivalis, most likely due to antagonism by the gingipain proteinase virulence factors. This study advances our understanding of how IL-36γ may promote oral mucosal immunity and tissue homeostasis, and how this may be dysregulated by bacterial pathogens.

Regulation of the Peptidoglycan Amidase PGLYRP2 in Epithelial Cells by Interleukin-36γ.

Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

MEK-ERK signaling diametrically controls the stimulation of IL-23p19 and EBI3 expression in epithelial cells by IL-36γ.

Interleukin (IL)-36 cytokines are important regulators of mucosal homeostasis and inflammation. We previously established that oral epithelial cells strongly upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL-36γ stimulates the expression of the IL-12 cytokine family members, IL-23p19 and Epstein-Barr Virus-Induced Gene 3 (EBI3), by oral epithelial cells; their expression was also selectively stimulated by IL-36α. Notably, IL-23p19 and EBI3 expression was not stimulated by P. gingivalis, thus suggesting that their expression by the oral epithelium in response to P. gingivalis is likely to be mediated in an autocrine manner by IL-36γ. The IL-36γ-inducible expression of IL-23p19 and EBI3 was found to be diametrically regulated by the mitogen-activated protein kinase/extracellular signal regulated kinase (MEK)-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereby the activation of MEK-ERK signaling likely functions as a negative feedback mechanism to limit EBI3 expression. Furthermore, epidermal growth factor receptor (EGFR) signaling, which is important for mucosal homeostasis, was demonstrated to modulate, in a MEK-ERK-dependent manner, the stimulation of IL-23p19 and EBI3 expression by IL-36γ. IL-23p19 and EBI3 have recently been shown to heterodimerize to form the novel cytokine IL-39 and promote neutrophil expansion. EBI3 has been shown to also have IL-12 cytokine family independent functions (e.g. mediating IL-6 trans-signaling). Thus, this study not only advances our understanding of how IL-36 cytokines may control mucosal inflammation, but also establishes EGFR signaling as a potentially important modulator of IL-36 cytokine function.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a ß-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Additive effect of repeated bouts of individualized frequency whole body vibration on postural stability in young adults.

Whole body vibration (WBV) has been shown to improve force and power output as well as flexibility and speed, with improvements suggested to result from reduced electromechanical delays, improved rate of force development, and sensitivity of muscle spindles. Fixed frequency studies on postural control have been somewhat equivocal; however, individualized frequency protocols have shown promising results in other motor tasks. To assess this, 18 healthy young adults experienced three 4-minute WBV sessions with postural control assessed before vibration, after multiple exposures, and during recovery, with altered levels of sensory information available to the participants. Sway velocity, sway path length, and sway area were assessed in each environment. Study findings revealed that stability was impacted following WBV, with more challenging environments eliciting improvements persisting for 20 minutes. When the environment was less challenging, postural stability was impaired; however, the effects dissipated quickly (10-20 min). It was determined that exposure to individualized frequency WBV served to impair postural control when the challenge was low, but resulted in heightened stability when the overall challenge was high and vestibular information was needed for stability.