Does farm fungicide use induce azole resistance in Aspergillus fumigatus?

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus.

Transition metal ions induce carnosinase activity in PepD-homologous protein from Porphyromonas gingivalis.

Aminoacylhistidine dipeptidase (EC 3.4.13.3; also Xaa-His dipeptidase, carnosinase, or PepD) catalyzes the cleavage and release of an N-terminal amino acid, which is usually a neutral or hydrophobic residue, from an Xaa-His dipeptide or degraded peptide fragment. PepD enzyme is found extensively in prokaryotes and eukaryotes, and belongs to the metallopeptidase family M20, a part of the metallopeptidase H (MH) clan. Carnosine is a naturally occurring dipeptide (ß-alanyl-l-histidine) present in mammalian tissues that has protective functions in addition to anti-oxidant and free-radical scavenging roles. During bacterial infections, degradation of l-carnosine via carnosinase or PepD-like enzymes may enhance the destructive potential of bacteria, resulting in a pathological impact. This process has been proposed to act in an anti-oxidant manner in vivo. In the present study, the recombinant PepD protein encoded by Porphyromonas gingivalis TDC60 pepD was generated and biochemically characterized. In addition, a recombinant dipeptidase enzyme was found to function not only as an alanine-aminopeptidase, but also as a carnosinase. Furthermore, when carnosine was used as substrate for PepD, the transition metals, Mn(2+), Fe(2+), Co(2+), and Ni(2+) stimulated the hydrolyzing activity of rPepD with ß-alanine and l-histidine. Based on its metal ion specificity, we propose that this enzyme should not only be termed l-aminopeptidase, but also a carnosinase.

Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease.

Porphyromonas gingivalis is a black-pigmented asaccharolytic anaerobe and a major causative agent of periodontitis. Here, we report the complete genome sequence of P. gingivalis strain TDC60, which was recently isolated from a severe periodontal lesion in a Japanese patient.

Characterization of hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis: its cellular distribution, thioredoxin activity and role in heme utilization.

BACKGROUND: The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively. RESULTS: We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions. CONCLUSION: P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.

Functional analysis of the thioredoxin domain in Porphyromonas gingivalis HBP35.

Periodontitis is one of the most common oral diseases in humans. This caused by infection by the oral bacterium Porphyromonas gingivalis. Our strategy to prevent this infection is to establish a passive immunization system in which endogenous antibodies can be applied directly to neutralize virulent factors associated with this bacterium. We focused our attention on the P. gingivalis 35 kDa surface protein, or HBP35, since this protein is involved not only in the coaggregation with oral miroflora but also in hemin binding. In addition, nucleotide sequencing of the gene, hbp35, coding for this protein revealed the presence of a catalytic center for thioredoxin, and we further attempted to characterized the protein by amino acid substitution. A total of four Cys residues were substituted for Ser residues by combining the simple method for site-directed mutagenesis and the heterodimer system, an approach designed to construct chimeric plasmids readily. Native and mutagenized hbp35 were introduced into the Eschericha coli dsbA mutant strain, JCB 572, defective in both alkaline phosphatase and motile activities due to inefficient disulfide bond formation. Transformant harboring the native hbp35 could complement the dsbA mutation, suggesting a role of disulfide bond formation of this protein in P. gingivalis cells. Possible roles of the Cys residues in complementation are discussed.

The r40-kDa outer membrane protein human monoclonal antibody protects against Porphyromonas gingivalis-induced bone loss in rats.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We recently reported the construction and characterization of human immunoglobulin G isotype clones, which were specifically reactive with recombinant (r) 40-kDa outer membrane protein (OMP) of P. gingivalis. The aim of this study was to investigate the efficacy of human monoclonal antibody (hMAb) against r40-kDa OMP of P. gingivalis to the protection alveolar bone loss by P. gingivalis in rats. METHODS: The role of 40-kDa OMP in the adherence of P. gingivalis to human gingival epithelial cells (HGECs) was examined by preincubating with r40-kDa OMP hMAb before adding the HGECs. Moreover, we used a rat model to examine the effect of the anti-r40-kDa OMP hMAb in alveolar bone loss by oral infection. Forty-six days after the last infection, the periodontal bone level was assessed morphometrically on defleshed rat jaws. RESULTS: The adherence to HGECs was reduced by 84% compared to adherence levels without the antibody. P. gingivalis could not be detected from rats in a P. gingivalis-non-infected group and a group that was administered the anti-r40-kDa OMP hMAb. The bone loss in P. gingivalis-infected animals that were administered the anti-r40-kDa OMP hMAb was significantly lower than that of P. gingivalis-infected rats. CONCLUSIONS: Our results suggest that transchromosomic mouse-derived hMAb against r40-kDa OMP of P. gingivalis protects against periodontal bone loss. This newly constructed anti-r40-kDa OMP hMAb was used to protect against periodontal diseases caused by P. gingivalis infection.

Gene expression of MC3T3-E1 cells on fibronectin-immobilized titanium using tresyl chloride activation technique.

Fibronectin (FN) can be immobilized directly on titanium surfaces using tresyl chloride activation technique. The key advantage of tresyl chloride activation technique lies in its simplicity. In this study, we examined the cell attachment and gene expression of MC3T3-E1 cells on FN-immobilized titanium using GeneChip. Cells attached on FN-immobilized titanium at a higher rate than untreated titanium. FN altered the gene expression profile, whereby 62 genes were found to be up-regulated, while 56 genes were found to down-regulate to over twice the level on day 14. FN not only enhanced the expression levels of IBSP and OMD, but also decreased SULF1 mRNA level. Taken together, the immobilization of FN on tresylated titanium promoted early matrix mineralization and bone formation.

Proinflammatory gene expression in mouse ST2 cell line in response to infection by Porphyromonas gingivalis.

Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.

An inhibitory effect of A20 on NF-kappaB activation in airway epithelium upon influenza virus infection.

Influenza is a major disease in humans. The reemergence of avian influenza A viruses has indicated that hyperinflammatory responses are closely related to the severity of disease. Influenza virus infection induces nuclear transcription factor kappaB (NF-kappaB) activation. NF-kappaB and NF-kappaB-dependent gene products promote lung inflammation and injury. Therefore, it is important to investigate the means to attenuate NF-kappaB activation. A20 is a cytoplasmic zinc finger protein that inhibits NF-kappaB activity, However, little is known about the role of A20 in influenza virus infection. Here, we have examined the role of A20 in influenza virus infection-induced NF-kappaB promoter activation in human bronchial epithelial cells. The results showed that (1) A20 protein and mRNA are inducible and expressed in the lung from mice and human bronchial epithelial cells upon influenza virus infection; (2) NF-kappaB promoter activation was induced in bronchial epithelial cells upon influenza virus infection; and (3) overexpression by transient transfection of A20 attenuated NF-kappaB promoter activation in bronchial epithelial cells. These results indicate that A20 may function as a negative regulator of NF-kappaB-mediated lung inflammation and injury upon influenza virus infection, thereby protecting the host against inflammatory response to influenza virus infection.

Production of a single-chain variable fraction capable of inhibiting the Streptococcus mutans glucosyltransferase in Bacillus brevis: construction of a chimeric shuttle plasmid secreting its gene product.

Periodontitis and dental caries are common oral diseases, in these days, and the passive immunization is one of the most effective approaches for prevention. For this purpose, we have constructed mouse and human monoclonal antibodies to inhibit the Porphyromonas gingivalis-associated hemagglutination and coaggregation. In addition, an artificial antibody, single-chain variable fraction, or scFv, which also inhibited the hemagglutination, was constructed. Specifically for dental caries, mouse and human monoclonal antibodies that inhibited the glucosyltransferase (GTF) activity, responsible for biofilm formation, were also constructed. The advantage of scFv over the native antibody is that the former molecule does not induce possible side-effects due to Fc, such as autoimmune disease, because it consists only of variable regions originating from both heavy and light chains. To increase the abilities of the antibody preparations, we attempted to construct an additional scFv using Bacillus brevis, a secretion-proficient gram-positive bacterium, as a host cell. An scFv protein possessing the same biological activity as that of the parental antibody was successfully secreted from a B. brevis transformant following the construction of a chimeric shuttle plasmid, which was accomplished by employing a new heterodimer system.

The trans-chromosomic mouse-derived human monoclonal antibody promotes phagocytosis of Porphyromonas gingivalis by neutrophils.

BACKGROUND: As a safe immunotherapeutic approach, human monoclonal antibody (hMAb) may be effective in clearing periodontopathic bacteria. The trans-chromosomic (TC) technology has recently been applied to construction of the TC mouse, which enables us to incorporate entire human chromosome fragments containing immunoglobulin (Ig) gene cluster. The aim of this study is to establish TC mouse-derived hMAb, and to test the in vitro opsonophagocytic activity. METHODS: Human Ig-producing TC mouse was immunized by recombinant 40-kDa outer membrane protein (r40-kDa OMP) of Porphyromonas gingivalis 381, and the spleen cells were fused with the mouse myeloma cell line. The specificity of antir40- kDa OMP hMAb was evaluated with the enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assays. Flow cytometric analyses were performed to assess the opsonophagocytic activity. RESULTS: We successfully constructed 99 IgG isotype clones (IgG1: 84; IgG2: 11; IgG4: four clones), which were specifically reactive with r40-kDa OMP. The anti-r40-kDa OMP IgG1 hMAbs promoted phagocytosis of P. gingivalis by neutrophils. Futhermore, an increased opsonophagocytic activitity of anti-r40-kDa OMP IgG1 hMAbs was observed not only in P. gingivalis 381, but also in the W50, W83, and Su63 strains. CONCLUSION: Our results document the TC mouse-derived hMAb to promote neutrophil phagocytosis of P. gingivalis, suggesting an immunotherapeutic option for clearance of P. gingivalis.

Human monoclonal antibody inhibits Porphyromonas gingivalis hemagglutinin activity.

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of chronic periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. One potential virulence factor, hemagglutinin, may mediate bacteria attachment onto and penetration into host cells, as well as agglutinate and lyse erythrocytes to intake heme, an absolute requirement for growth. We previously cloned the gene encoding the 130 kDa hemagglutinin domain (130k HMGD) and identified its functional domain. The construction of a human monoclonal antibody that is capable of inhibiting the hemagglutinating ability is significant and important toward the development of passive immunotherapy. METHODS: Human lymphocytes isolated from a donor, who had high antibody titer against the recombinant 130k HMGD (r130k HMGD), were immortalized by Epstein-Barr virus, and specific antibody-producing B cells were established by panning using the r130k HMGD. RESULTS: The constructed HuMAb-HMGD1, IgG subclass, recognized the r130k HMGD as well as the 43 and 49 kDa major bands in P. gingivalis cells and vesicles. The HuMAb-HMGD1 significantly inhibited hemagglutinating activity of P. gingivalis vesicles in a dose-dependent manner. Furthermore, the HuMAb-HMGD1 recognized the synthetic peptide, EGSNEFAPVQNLTGSSVG, which contains the functional domain of 130k HMGD. CONCLUSION: The newly constructed HuMAb-HMGD1 may prove to be useful for the development of passive immunization against periodontal diseases caused by P. gingivalis infection, pending the results of fertility study in disease mode.

Production of antibody against a synthetic peptide of Porphyromonas gingivalis 40-kDa outer membrane protein.

Porphyromonas gingivalis has been implicated as a major pathogen in periodontal diseases. We previously cloned a 40-kDa outer membrane protein (OMP) gene from P. gingivalis 381 and succeeded in producing sufficient quantities of the recombinant protein for experimental use. Since antibodies against the recombinant (r) 40-kDa OMP have potent ability to kill P. gingivalis cells by complement activation and opsonization, r40-kDa OMP has been the subject of considerable interest as a possible vaccine candidate. In this study, in order to develop a component vaccine, the immunodominant domain in 40-kDa OMP was identified. Peptides corresponding to portions of the N-terminal regions of 40-kDa OMP were synthesized chemically and their immunoreactivities with antibody against r40-kDa OMP were tested. The 16-mer peptide, LDDEYKERVFQTFVHY, was found to react strongly with the antibody. Furthermore, a rabbit antibody was prepared by immunization with the 16-mer peptide, cross-linked with dehydrofolate reductase, and its immunoreactivity was then examined. In a BIAcore experiment the antibody clearly reacted with r40-kDa OMP as well as P. gingivalis strains. These findings suggest that the 16-mer synthetic peptide may be useful for development of a component vaccine against P. gingivalis.

Identification of an O-antigen chain length regulator, WzzP, in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN_2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN_2005 mutant were shorter than those of the wild type; (ii) the PGN_2005 protein was located in the inner membrane fraction; and (iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN_2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN_1896 and PGN_1233 proteins and the PGN_1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively.

Acute application of cisplatin affects methylation status in neuroblastoma cells.

The pharmacological mechanism of the anti-cancer effect of cisplatin is well known to be DNA intercalation, but the direct or indirect effects of cisplatin on protein expression in cancer cells remain to be explained. In this study, we used a proteomic approach to clarify the early impact of cisplatin on protein expression. In a 2-dimensional gel electrophoresis proteomic experiment, the application of cisplatin for 24 h increased the expression of four proteins and decreased the levels of one protein in neuroblastoma IMR-32 cells. Levels of S-adenosyl-L-homocysteine hydrolase, a key enzyme in methylation metabolism, were increased the most. Therefore, we examined the methylation status of histone proteins. Histone H3K9 methylation was reduced by the application of cisplatin for 24 h. These results suggest that acute cisplatin treatment alters methylation status. Thus, these data can help clarify the unknown pharmacological mechanisms of cisplatin, including the anticancer effect, adverse effects and/or the mechanism of drug resistance.

A monoclonal antibody against fimA type II Porphyromonas gingivalis inhibits IL-8 production in human gingival fibroblasts.

The periodontal pathogen Porphyromans gingivalis is classified into six groups (types I-V and Ib) based on the genotype of the fimbriae A (fimA) gene. Among genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. The present study was undertaken to develop passive immunotherapy monoclonal antibodies (MAbs) against periodontitis, which are capable of inhibiting virulency and were constructed through the immunization of outer membrane vesicles (OMV) fraction of fimAII strain, TDC60, using mouse hybridoma technology. MAbs that recognized OMV by ELISA assay were identified, and 28 clones were screened by Western blot analysis. After purifying these MAbs using protein G column, the effect of the MAb on IL-8 production from human gingival fibroblasts by OMV was examined. We selected MAb TDC4-33H, which strongly inhibited the IL-8 production with a higher MAb production rate. Since the MAb showed an individual ladder-like profile against OMV by Western blotting, we further examined the reactivity against lipopolysaccharides (LPS) from TDC60, W83 (fimAIV), and ATCC33277 (fimAI). As a result, MAb TDC4-33H recognized all LPSs. Moreover, MAb TDC4-33H significantly inhibited the LPS-stimulated IL-8 production in human gingival fibroblasts. These findings suggest that MAb TDC4-33H reacts with LPS and may be useful for passive immunotherapy through neutralizing IL-8 production in gingival fibroblasts by P. gingivalis LPS.

Oxidative stress induces phosphorylation of the ABC transporter, ATP-binding protein, in Porphyromonas gingivalis.

The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of periodontal disease. Although P. gingivalis is an anaerobic bacterium, it exhibits aerotolerance and can survive in periodontal pockets, indicating that it must possess a mechanism for protection against oxidative stress, although the precise details are still unclear. Recently, phosphorylation signaling has been implicated in the regulation of bacterial virulence. In the present study, to examine the effect of oxidative stress on phosphorylation of proteins in P. gingivalis, we analyzed oxidative stress-induced alterations of phosphorylated proteins using two-dimensional electrophoresis with phosphoprotein staining coupled with MALDI-TOF mass spectrometry analysis. Among the phosphorylated proteins analyzed, we identified an increase in phosphorylation of the ABC transporter, ATP-binding protein (PG0258). Since the ABC transporter family is known to be involved in lipopolysaccharide (LPS) biosynthesis, we examined the level of LPS using an endotoxin assay and found that LPS production was increased in P. gingivalis. Our present findings suggest that the early response of P. gingivalis to oxidative stress could trigger the development and progression of periodontal disease through enhancement of LPS production by phosphorylation of the ABC transporter, ATP-binding protein.

Monoclonal antibodies produced against lipopolysaccharide from fimA Type II Porphyromonas gingivalis.

An important periodontal pathogen, Porphyromans gingivalis strains are classified into six genotypes (types I-V and Ib), based on the genotype of the fimbriae A (fimA). Among the genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. To develop passive immunotherapy, over 300 hybridoma clones were constructed through immunization of cell extracts of fimA type II strain P. gingivalis TDC60 using hybridoma technology. Among these clones, 15 MAbs recognized TDC60 lipopolysaccharide (LPS) with an individual ladder-like structure by Western blot analysis. Further Western blotting of the 15 MAbs against LPS from TDC60, FDC381 (fimA type I), and W83 (fimA type IV) of P. gingivalis and Escherichia coli was carried out. None of these MAbs recognized E. coli LPS, and divided into at least three different Western blot patterns. To confirm the specificity to LPS, three clones were selected and competition assays were carried out using TDC60 LPS. All three MAbs reduced the reactivity against TDC60 LPS after absorption of the LPS in a dose-dependent manner. These findings suggest that MAbs recognizing different epitopes of P. gingivalis LPS were successfully constructed, and these MAbs may be useful in neutralizing P. gingivalis infection.

Effects of linear polarized infrared light irradiation on the transcriptional regulation of IL-8 expression in IL-1beta-stimulated human rheumatoid synoviocytes involves phosphorylation of the NF-kappaB RelA subunit.

Although recent clinical studies have shown that laser therapy acts as an anti-inflammatory effector in the treatment of some diseases, little is known about the mechanism by which it acts in rheumatoid arthritis (RA) patients. The purpose of our work was to examine how irradiation with linear polarized infrared light (LPIL) suppresses inflammatory responses in the MH7A rheumatoid fibroblast-like synoviocyte cell line. We initially confirmed the effects of two disease-modifying anti-rheumatic treatments, LPIL irradiation and dexamethasone (Dex) administration, under experimental inflammatory conditions using gene chip technology. We found that LPIL exerted a smaller effect on gene transcription than Dex; however, IL-1beta-inducible target genes such as the CXCL type chemokines IL-8, IL-1beta and IL-6 were all clearly suppressed by LPIL to the same degree as by Dex. We also found that IL-1beta-induced release of IL-8 from MH7A cells was completely blocked by pretreatment with the (IL-8) inhibitor Bay11-7085, indicating that activation of NF-kappaB signaling plays an important role in the secretion of IL-8. Although the levels of NFKB1 and RELA transcription were unaffected by IL-1beta stimulation, phosphorylation of RelA S276 was suppressed by both LPIL and Dex. Thus LPIL likely exerts its anti-inflammatory effects by inhibiting the release of the inflammatory chemokine IL-8. A fuller understanding of the anti-inflammatory mechanism of LPIL in rheumatoid synoviocytes could serve as the basis for improved treatment of RA patients in the future.