Th17 cells differentiated with mycelial membranes of Candida albicans prevent oral candidiasis.

Candida albicans is a human commensal that causes opportunistic infections. Th17 cells provide resistance against mucosal infection with C. albicans; however, the T cell antigens remain little known. Our final goal is to find effective T cell antigens of C. albicans that are responsible for immunotherapy against candidiasis. Here, we prepared fractions including cytosol, membrane and cell wall from yeast and mycelial cells. Proteins derived from a membrane fraction of mycelial cells effectively induced differentiation of CD4+ T cells into IL-17A-producing Th17 cells. To confirm the immunological response in vivo of proteins from mycelial membrane, we performed adoptive transfer experiments using ex vivo stimulated CD4+ T cells from IL-17A-GFP reporter mice. Mycelial membrane-differentiated CD4+ Th17 cells adoptively transferred intravenously prevented oral candidiasis by oral infection of C. albicans, compared with control anti-CD3-stimulated CD4+ T cells. This was confirmed by the clinical score and the number of neutrophils on the infected tissues. These data suggest that effective T cell antigens against candidiasis could be present in the membrane protein fraction of mycelial cells. The design of novel vaccination strategies against candidiasis will be our next step.

ATPase activity regulation by leader peptide processing of ABC transporter maturation and secretion protein, NukT, for lantibiotic nukacin ISK-1.

Lantibiotic nukacin ISK-1 is produced by Staphylococcus warneri ISK-1. The dual functional transporter NukT, an ABC transporter maturation and secretion protein, contributes to cleavage of the leader peptide from the prepeptide (modified NukA) and the final transport of nukacin ISK-1. NukT consists of an N-terminal peptidase domain (PEP), a C-terminal nucleotide-binding domain (NBD), and a transmembrane domain (TMD). In this study, NukT and its peptidase-inactive mutant were expressed, purified, and reconstituted into liposomes for analysis of their peptidase and ATPase activities. The ATPase activity of the NBD region was shown to be required for the peptidase activity of the PEP region. Furthermore, we demonstrated for the first time that leader peptide cleavage by the PEP region significantly enhanced the ATPase activity of the NBD region. Taken together, the presented results offer new insights into the processing mechanism of lantibiotic transporters and the necessity of interdomain cooperation.

Antifungal activity in vitro and in vivo of a salmon protamine peptide and its derived cyclic peptide against Candida albicans.

Protamine peptide (PP) derived from salmon is a 14-mer with 10 arginine residues. We investigated the in vitro and in vivo antifungal activity of PP against Candida albicans PP showed a concentration-dependent dual mode of action, with fungicidal activity and inhibitory activity for hyphal development in vitro. At lethal concentrations of PP, intracellular accumulation of PP was energy-dependent but independent of endocytosis, and resulted in ATP efflux and the generation of reactive oxygen species in the cells. PP at sublethal concentrations inhibited hyphal development in C. albicans by binding to the cell surface. Though antifungal activity of PP was inactivated by high concentrations of NaCl, the antifungal activity of the synthetic cyclic (via a disulfide bond) form of PP (cyclic PP) was not. Cyclic PP also showed the concentration-dependent dual mode of action, and had five-fold greater antifungal activity than PP. The advantage of antifungal activity of cyclic PP compared with PP in vitro resulted in a high in vivo efficacy in a murine oral candidiasis model. Oral treatment with cyclic PP inhibited hyphal development of C. albicans on mouse tongues and protected against the development of severe candidiasis. This study shows the therapeutic potential of cyclic PP as an antifungal peptide against C. albicans.

Bactericidal activity of nukacin ISK-1: an alternative mode of action.

We previously reported bacteriostatic action of nukacin ISK-1 against Bacillus subtilis JCM 1465(T). Here, we found its bactericidal activity against Micrococcus luteus DSM 1790 and Staphylococcus simulans 22, showing decrease in cell viability, cell lysis, and dissipation of the membrane potential. Moreover, leakage of small molecules such as K(+), suggested the formation of small-sized or specific K(+)-conducting-pores by nukacin ISK-1.

Mobility gene expression differences among wild-type, Mmp20 null and Mmp20 over-expresser mice plus visualization of 3D mouse ameloblast directional movement.

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20-/- mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20-/- and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20-/- mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.

Dispensable Role of Aire in CD11c+ Conventional Dendritic Cells for Antigen Presentation and Shaping the Transcriptome.

Aire, the defect of which is responsible for the development of autoimmunity, is predominantly expressed in medullary thymic epithelial cells, and it controls a wide variety of genes, including those of tissue-restricted Ags, for establishing thymic tolerance. Aire is also expressed from APCs in the periphery, called extrathymic Aire-expressing cells (eTACs), and their complementing role to thymic tolerance has been suggested. eTACs are composed of two distinct classes of APCs, conventional dendritic cell (cDC)-type and group 3 innate lymphoid cell (ILC3)-like-type expressing retinoic acid receptor-related orphan receptor γt (RORγt). Although the essential role of Aire in the latter in the Th17-mediated immune response against Candida albicans has been reported, the role of Aire in the cDC-type eTACs for this action has not been examined. Furthermore, the significance of Aire in the production of the transcriptome of the cDC-type eTACs remains unknown. We have approached these issues using a high-fidelity Aire-reporter mouse strain. We found that although the cDC-type eTACs dominated ILC3-like-type eTACs in number and they served as efficient APCs for the immune response against an exogenous Ag as well as for the C. albicans-specific Th17 immune response, loss of Aire in cDC-type eTACs showed no clear effect on these functions. Furthermore, loss of Aire showed no major impact on the transcriptome from cDC-type eTACs. These results suggested that Aire in cDC-type eTACs may not have a cell-intrinsic role in the immune response in contrast to the role of Aire in ILC3-like-type eTACs.

Lantibiotic transporter requires cooperative functioning of the peptidase domain and the ATP binding domain.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn(106) is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT.

Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic.

Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 µM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis.

Antimicrobial mechanism of lantibiotics.

Lantibiotics are ribosomally synthesized antimicrobial peptides that commonly target the cell wall precursor lipid II during their antimicrobial mechanism and exert their inhibitory activity by (i) inhibition of cell wall biosynthesis, and (ii) stable pore formation in the target membrane. Type-A(I) (i.e. nisin) and two-component (i.e. lacticin 3147) lantibiotics initially interact with lipid II to stabilize the complex, which then proceeds to inhibit cell wall biosynthesis and pore formation. Type-A(II) (i.e. nukacin ISK-1) and type-B (i.e. mersacidin) lantibiotics also use lipid II as a docking molecule, but can only inhibit cell wall biosynthesis without forming pores. In the present paper, we review the antimicrobial mechanism of different types of lantibiotics, their current progress and future prospect.

Candida albicans Msi3p, a homolog of the Saccharomyces cerevisiae Sse1p of the Hsp70 family, is involved in cell growth and fluconazole tolerance.

We investigated the cellular function of Msi3p, belonging to the heat shock protein 70 family, in Candida albicans. The mutant strain tetMSI3 was generated, in which MSI3 was controlled by a tetracycline-repressive promoter, because there is evidence to suggest that MSI3 is an essential gene. We controlled the MSI3 expression level by doxycycline (DOX) and compared its phenotype with that of a control strain with the tetracycline-repressive promoter and a wild-type copy MSI3. The results indicated that MSI3 was essential for cell growth. In addition, all the tetMSI3-infected mice survived after DOX administration. Drug susceptibility tests indicated that repression of MSI3 expression resulted in hypersensitivity to fluconazole and conferred fungicidal activity to fluconazole. The expression levels of MSI3 and calcineurin-dependent genes were upregulated in response to fluconazole in the control strain. In tetMSI3, the upregulation of MSI3 was lost, and the expression level of the calcineurin-dependent genes was no longer elevated in response to fluconazole and was not affected by DOX, indicating that the upregulation of MSI3 expression was required for the induction of the calcineurin-dependent gene expression. These data suggest that Msi3p confers fluconazole tolerance by partially influencing the calcineurin signaling pathway and also other tolerance mechanisms.

Enhanced production of nukacin D13E in Lactococcus lactis NZ9000 by the additional expression of immunity genes.

Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation.

In vitro efficacy of continuous mild heat stress on the antifungal susceptibility of Candida albicans biofilm formation.

Elevation in the temperature induces heat stress to both host cells and the invading pathogen. This study aimed to determine whether continuous mild heat stress (increased temperature without causing significant damage to host cells) can increase susceptibility of biofilm formation of the opportunistic fungal pathogen Candida albicans to low concentrations of three typical antifungal agents. In this way the side effects associated with higher concentrations of the antifungal agents on host cells would be reduced. Fluconazole and micafungin at concentrations ranging from 0.0625 to 2 µg/mL and amphotericin B at concentrations ranging from 0.0625 to 1 µg/mL inhibited less than 20% of cells in biofilm formation. Biofilm formation at 39 or 41°C compared to 37°C resulted in increased susceptibility to the three agents, but especially micafungin. These data suggest that mild heat stress (39°C) would be valuable for increasing the effectiveness of low concentrations of antifungal agents against C. albicans biofilm formation. Thus, the concept of continuous mild heat stress at the site of insertion of medical devices or catheters combined with antifungal agents could be beneficial.

Antibacterial peptides "bacteriocins": an overview of their diverse characteristics and applications.

Bacteriocins are ribosomally synthesized antibacterial peptides produced by bacteria that inhibit the growth of similar or closely related bacterial strains. A number of bacteriocins from a wide variety of bacteria have been discovered, and their diverse structures have been reported. Growing evidence suggests that bacteriocins have diverse structures, modes of action, mechanisms of biosynthesis and self-immunity, and gene regulation. Bacteriocins are considered as an attractive compound in food and pharmaceutical industries to prevent food spoilage and pathogenic bacterial growth. Furthermore, elucidation of their biosynthesis has led to the use of bacteriocin-controlled gene-expression systems and the biosynthetic enzymes of lantibiotics, a class of bacteriocins, as tools to design novel peptides. In this review, we summarize and discuss currently known information on bacteriocins produced by Gram-positive bacteria and their applications.

Pathobiont-responsive Th17 cells in gut-mouth axis provoke inflammatory oral disease and are modulated by intestinal microbiome.

Host immune response via Th17 cells against oral pathobionts is a key mediator in periodontitis development. However, where and how the Th17-type immune response is induced during the development of periodontitis is not well understood. Here, we demonstrate that gut translocation of the oral pathobiont Porphyromonas gingivalis (Pg) exacerbates oral pathobiont-induced periodontitis with enhanced Th17 cell differentiation. The oral pathobiont-responsive Th17 cells are differentiated in Peyer's patches and translocated systemically in the peripheral immune tissues. They are also capable of migrating to and accumulating in the mouth upon oral infection. Development of periodontitis via the oral pathobiont-responsive Th17 cells is regulated by the intestinal microbiome, and altering the intestinal microbiome composition with antibiotics affects the development of periodontitis. Our study highlights that pathobiont-responsive Th17 cells in the gut-mouth axis and the intestinal microbiome work together to provoke inflammatory oral diseases, including periodontitis.

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1.

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue.

Bacterial-induced maternal interleukin-17A pathway promotes autistic-like behaviors in mouse offspring.

Maternal immune activation (MIA) by an infection is considered to be an important environmental factor of fetal brain development. Recent animal model on MIA induced by polyinosinic:polycytidylic acid, a mimic of viral infection, demonstrates that maternal IL-17A signaling is required for the development of autism spectrum disorder (ASD)-like behaviors of offspring. However, there is little information on bacterial infection. In this study, we aim to elucidate the influence of MIA induced by lipopolysaccharide (LPS) to mimic a bacterial infection on fetal brain development. We demonstrated that LPS-induced MIA promoted ASD-like behaviors in mouse offspring. We further found that LPS exposure induced acute phase immune response: elevation of serum IL-17A levels in MIA mothers, upregulation of Il17a mRNA expression and increase of IL-17A-producing γδ T cells in the uterus, and upregulation of Il17ra mRNA expression in the fetal brain. Blocking of IL-17A in LPS-induced MIA ameliorated ASD-like behaviors in offspring. Our data suggest that bacterial-induced maternal IL-17A pathway promotes ASD-like behaviors in offspring.

Nukacin ISK-1, a bacteriostatic lantibiotic.

We determined the mode of action of nukacin ISK-1. It did not cause membrane potential dissipation or the efflux of ATP or K(+) ions from the cells of a sensitive bacterial strain; however, it blocked the membrane depolarization activity of nisin. Nukacin ISK-1-treated cells had single arrangements of cells without the formation of a complete septum. A remarkable reduction in cell wall width was also observed, but cytoplasmic content was unaffected. We concluded that nukacin ISK-1 is bacteriostatic.

Mild Heat Stress Affects on the Cell Wall Structure in Candida albicans Biofilm.

We previously reported that Candida albicans responded to mild heat stress in a range of temperature elevations simulating fever, and concluded that mild heat stress increases susceptibility to antifungal drugs. In this study, we show that mild heat stress causes a morphological change in hyphae during the process of biofilm formation. We found that mild heat stress extended the period of hyphal stage maintenance in C. albicans biofilm. Although the rate of hyphal change from yeast form to hyphal form reached the maximum within 3 hr, later, almost every cell quickly reverted to the yeast growth phase within 6 hr at 37°C but not at 39°C, or under mild heat stress. Electron microscopy using a smart specimen preparation technique revealed that mild heat stress significantly increased the thickness of the inner cell wall accompanied by a decrease in density of the outer cell wall in the hyphae of C. albicans biofilm. To identify the gene responsible for the morphological changes associated with mild heat stress, we performed microarray gene expression analysis. Eleven genes were upregulated and 17 genes were downregulated under mild heat stress in biofilm cells. The increased PHR1 gene expression in response to mild heat stress was confirmed in quantitative RT-PCR analysis. The mutant upregulated PHR1 expression showed the same sensitivity against antifungal drug micafungin as dependent on mild heat stress. Our findings point to possible therapeutic effects of hyperthermia as well as to the effect of fever during infections.

Binding specificity of the lantibiotic-binding immunity protein NukH.

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin(4-27) and T24A nukacin(4-27), respectively) to nukH-expressing cells were considerably lower than those of nukacin(4-27), suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin(4-27), a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells.

The lantibiotic nukacin ISK-1 exists in an equilibrium between active and inactive lipid-II binding states.

The lantibiotic nukacin ISK-1 exerts antimicrobial activity through binding to lipid II. Here, we perform NMR analyses of the structure of nukacin ISK-1 and the interaction with lipid II. Unexpectedly, nukacin ISK-1 exists in two structural states in aqueous solution, with an interconversion rate on a time scale of seconds. The two structures differ in the relative orientations of the two lanthionine rings, ring A and ring C. Chemical shift perturbation induced by the titration of lipid II reveals that only one state was capable of binding to lipid II. On the molecular surface of the active state, a multiple hydrogen-bonding site formed by amino acid residues in the ring A region is adjacent to a hydrophobic surface formed by residues in the ring C region, and we propose that these sites interact with the pyrophosphate moiety and the isoprene chain of the lipid II molecule, respectively.