Hiromi Nishi

Reduced Content of Lysyl-Phosphatidylglycerol in the Cytoplasmic Membrane Affects Susceptibility to Moenomycin, as Well as Vancomycin, Gentamicin, and Antimicrobial Peptides, in Staphylococcus aureus

ABSTRACT An association between moenomycin resistance and vancomycin intermediate resistance in Staphylococcus aureus was demonstrated previously. Thus, to elucidate the mechanism of vancomycin intermediate resistance, we searched for factors contributing to moenomycin resistance. Random Tn551 insertional mutagenesis of methicillin-resistant S. aureus strain COL yielded three mutants with decreased susceptibilities to moenomycin. Correspondingly, these mutants also exhibited slightly decreased susceptibilities to vancomycin. Genetic analysis revealed that two of the mutants had Tn551 insertions in the fmtC (mprF) gene, which is associated with the synthesis of lysyl-phosphatidylglycerol. The third Tn551 insertion was located in the lysC gene, which is involved in the biosynthesis of lysine from aspartic acid. Consequently, mutations in both of these loci reduced the lysyl-phosphatidylglycerol content in the cell membrane, giving it a more negative net charge. The positively charged antibiotic gentamicin and cationic antimicrobial peptides such as β-defensins and CAP18 were more effective against the mutants. The levels of moenomycin and vancomycin binding to intact cells was also greater in the mutants than in the wild type, while the binding affinity was not altered when cells boiled in sodium dodecyl sulfate were used, indicating that both agents had higher affinities for the negatively charged membranes of the mutants. Therefore, the membrane charge of S. aureus appears to influence the efficacies of moenomycin, vancomycin, and other cationic antimicrobial agents.

The gate controlling cell wall synthesis in Staphylococcus aureus

Glucosamine‐6‐P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine‐6‐P is processed sequentially to UDP‐N‐acetylglucosamine, while to enter the glycolysis pathway, glucosamine‐6‐P is isomerized by NagB to fructose‐6‐P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine‐6‐P formation from N‐acetylglucosamine‐6‐P, and GlmS in that from fructose‐6‐P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N‐acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N‐acetylglucosamine. This and the preferential incorporation of N‐acetylglucosamine over glucose into cell wall material showed that N‐acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.

Increased resistance to cationic antimicrobial peptide LL-37 in methicillin-resistant strains of Staphylococcus aureus.

OBJECTIVES The susceptibility of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), to host-derived cationic antimicrobial peptides was investigated. METHODS We examined the susceptibility of 190 clinical strains of methicillin-susceptible S. aureus (MSSA) and 304 strains of MRSA to two different classes of cationic antimicrobial peptides: LL-37 and human beta-defensin-3 (hBD3). Out of the total 494 clinical strains, a random selection of 54 S. aureus strains was examined to establish the relationship between the net charge, or zeta potential, of each strain and its susceptibility to hBD3 or LL-37. To further confirm bacterial susceptibility to either hBD3 or LL-37, we concurrently measured: (i) percentage survival after in vitro bacterial exposure and (ii) MBCs for both MRSA and MSSA strains. RESULTS Of the 54 randomly selected S. aureus strains, those MRSA strains resistant to LL-37 showed significantly higher zeta potentials than those susceptible to LL-37 (P < 0.05). In contrast, there was no difference in bacterial zeta potentials for MRSA strains that showed either resistance or susceptibility to hBD3. In addition, resistance to LL-37, but not to hBD3, as determined by either percentage survival or MBC, was significantly elevated in highly methicillin-resistant strains of S. aureus when compared with MSSA strains (P < 0.01). CONCLUSIONS Clinical strains of MRSA, but not MSSA, that demonstrated an increased net charge also showed elevated resistance to LL-37, but not to hBD3.

Regulation of CXCL9/10/11 in Oral Keratinocytes and Fibroblasts

Th1 and Th2 cytokines such as interferon-γ (IFN-γ ) , tumor necrosis factor- α (TNF-α ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN-γ showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF-α caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN-γ in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-γ and TNF-α in GT1 cells. Thus, IFN-γ , TNF-α , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.

Interleukin-8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll-like receptors.

Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll‐like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1–10. Interleukin‐8 (IL‐8) production by RT7 cells was induced by treatment with TLRs 1–9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL‐8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor‐alpha‐induced IL‐8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.

A Case of SAPHO Syndrome With Diffuse Sclerosing Osteomyelitis of the Mandible Treated Successfully With Prednisolone and Bisphosphonate

*Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. †Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ‡Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. §Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ¶Department of Clinical Immunology and Rheumatology, Hiroshima University Hospital, Hiroshima, Japan. #Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. **Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ††Department of Surgery, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ‡‡Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Address correspondence and reprint requests to Dr Shigeishi: Department of Oral and Maxillofacial Surgery, Division of CervicoGnathostomatology, Graduate School of Biomedical Sciences, Hirod

Moenomycin-resistance is associated with vancomycin-intermediate susceptibility in Staphylococcus aureus.

We have previously isolated a vancomycin‐intermediate susceptibility mutant from methicillin‐resistant Staphylococcus aureus (MRSA) strain COL, and demonstrated the increased glycan‐chain length and the decreased moenomycin‐susceptibility. To further investigate the relationship between the resistance to vancomycin and to moenomycin, we isolated moenomycin‐resistant mutants (4–16 fold higher compared to the parent) from 5 MRSA and 2 methicillin‐sensitive S. aureus (MSSA) strains. The MRSA mutants showed a decreased susceptibility to vancomycin (2–4 fold), teicoplanin (2–4 fold) and an increased susceptibility to methicillin (2–8 fold). MSSA strains also showed similar results with those of MRSA strains except that there was no alteration of methicillin susceptibility. Among the mutants, three mutants including two MRSA mutants and one MSSA mutant were analyzed by electron microscopy, and they showed thickened cell walls compared to those of the parents. The glycan‐chain length of the peptidoglycan of the mutant was shown to be slightly longer than that of the parent, but the muropeptide profile was very similar. The expression levels of all PBPs were similar to those of the parent. Furthermore, the nucleotide sequences of sgtA, sgtB and pbp2 in the mutant were identical to those of the parent. These results indicate that the moenomycin‐resistance is closely associated with vancomycin‐intermediate susceptibility in S. aureus.

Wound healing effects of gingival fibroblasts cultured in animal-free medium.

OBJECTIVE The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal-free medium (HFDM1). METHODS We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. RESULTS The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5-2% was added to HFDM1. Wound healing cytokines, including transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL-6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbeccos modified Eagles medium with 10% fetal calf serum. CONCLUSION Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.

CX3CL1 expression induced by Candida albicans in oral fibroblasts

Oral fibroblasts as well as keratinocytes are thought to influence host inflammatory responses against Candida albicans. However, little is known about chemokine expressions in oral fibroblasts against C. albicans infection. We therefore examined whether C. albicans induced several chemokines including fractalkine/CX3CL1 (CX3CL1), a unique chemokine that has properties of both chemoattractants and adhesion molecules, in fibroblasts and keratinocytes. The addition of C. albicans live cells to human immortalized oral keratinocytes (RT7) resulted in increases in the mRNA levels of multiple chemokines, but not of CX3CL1. In contrast, live and heat-killed C. albicans caused an increase in CX3CL1 mRNA and protein expression in human immortalized oral fibroblasts (GT1). CX3CL1 mRNA expression in GT1 cells was also enhanced by stimulation with a nonalbicans species of Candida. Further, the CX3CL1 chemokine domain showed antifungal activity against C. albicans. CX3CL1 secreted by oral fibroblasts appears to play an important role in the oral immune response to C. albicans infection.

TNF-α-induced IL-6 and MMP-9 expression in immortalized ameloblastoma cell line established by hTERT.

OBJECTIVE Ameloblastoma (AM) shows locally invasive behaviour. However, biological investigations regarding regulation of gene expression associated with AM pathological features are difficult to perform, because AM cells can be passaged for a few generations due to senescence. We report a newly established immortalized AM cell line, AMB cells, by transfection with human telomerase reverse transcriptase (hTERT). Furthermore, we examined whether TNF-α modulates bone resorption-related genes, IL-6 and MMP-9 in cooperation with TGF-β or IFN-γ. MATERIALS AND METHODS Following transfection of an hTERT expression vector into AM cells using a non-viral method, the effects of cytokines on the expressions of IL-6 and MMP-9 mRNA were examined using real-time PCR. TNF-α-induced NF-κB activity was examined by western blotting and transcription factor assays. RESULTS AMB cells continued to grow for more than 100 population doublings. Stimulation with TNF-α increased IL-6 and MMP-9 mRNA expressions, as well as NF-κB activation. Furthermore, TGF-β and IFN-γ dramatically increased TNF-α-mediated expressions of MMP-9 and IL-6 mRNA, respectively, while those responses were suppressed by NF-κB inhibitor. CONCLUSION We established an immortalized AM cell line by hTERT transfection. TNF-α-mediated regulation of MMP-9 and IL-6 via NF-κB may play an important role in the pathological behaviour of AMs, such as bone resorption.