Sakuo Yamada

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Phage conversion of exfoliative toxin A production in Staphylococcus aureus

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (φETA) that encodes ETA and determined the complete nucleotide sequence of the φETA genome. φETA has a head with a hexagonal outline and a non‐contractile and flexible tail. The genome of φETA is a circularly permuted linear double‐stranded DNA, and the genome size is 43 081 bp. Sixty‐six open reading frames (ORFs) were identified on the φETA genome, including eta, which was found to be located very close to a putative attachment site (attP). φETA converted ETA non‐producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that φETA or related phages are responsible for the acquisition of eta genes in S. aureus.

Pleiotropic Roles of Polyglycerolphosphate Synthase of Lipoteichoic Acid in Growth of Staphylococcus aureus Cells

Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus Delta ltaS mutant could grow at 30 degrees C but not at 37 degrees C. Even at the permissive temperature, Delta ltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of Delta ltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the Delta ltaS mutant at 37 degrees C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the Delta ltaS mutation was found to be synthetically lethal with the Delta tagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30 degrees C, suggesting that LTA and WTA compensate for one another in an essential function.

Identification and molecular characterization of an N-acetylmuramyl-l-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N‐acetylmuramyl‐l‐alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N‐acetylmuramyl‐l‐Ala bonds in dimeric cross‐bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.

Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans (Aa) is one of the pathogenic bacteria involved in periodontal diseases. We have previously identified six major outer membrane proteins (Omps) of Aa Y4. Among them is an Omp with high molecular mass, designated Omp100, which has homology to a variety of virulence factors. Electron microscopic observation indicated that Omp100 is randomly localized on the cell surface of Aa. Aa Y4 has been shown to adhere and invade KB or normal human gingival keratinocytes. Anti‐Omp100 antibody inhibited 50% of adhesion and 70% of invasion of Aa Y4 to KB cells. An Omp100 knock‐out mutant had a decreased adhesion and invasion efficiency of 60%, compared with that of the wild type. Escherichia coli HB101 expressing Omp100 adhered twofold and invaded 10‐fold more than the wild‐type E. coli HB101. HB101 expressing Omp100 showed resistance to serum by trapping factor H, an inhibitor for C3b, with Omp100. Omp100 induced inflammatory cytokine responses of interleukin (IL)‐8, IL‐6 and tumour necrosis factor (TNF)α in epithelial cells, and induced IL‐1β and TNFα production in mouse macrophages. These results indicate that Omp100 is a versatile virulence factor that may demonstrate potential significance in the onset of periodontal diseases related to Aa.

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.

Molecular Characterization of an atl Null Mutant of Staphylococcus aureus

atl is a gene encoding a bifunctional peptidoglycan hydrolase of Staphylococcus aureus. The gene product of atl is a 138 kDa protein that has an amidase domain and a glucosaminidase domain, and undergoes processing to generate two major peptidoglycan hydrolases, a 51 kDa glucosaminidase and a 62 kDa amidase in culture supernatant. An atl null mutant was isolated by allelic replacement and characterized. The mutant grew in clusters and sedimented when grown in broth culture. Analysis of peptidoglycan prepared from the wild type and the mutant revealed that there were no differences in muropeptide composition or in glycan chain length distribution. On the other hand, the atl mutation resulted in pleiotropic effects on cell surface nature. The mutant cells showed complete inhibition of metabolic turnover of cell wall peptidoglycan and revealed a rough outer cell wall surface. The mutation also decreased the amount of protein non‐covalently bound to the cell surface and altered the protein profile, but did not affect proteins covalently associated with the cell wall. Lysis of growing cells treated with otherwise lytic concentration of penicillin G was completely inhibited in the mutant, but that of non‐growing cells was not affected by the mutation. The atl mutation did not significantly affect the ability of S. aureus to provoke an acute infection when inoculated intraperitoneally in a mouse sepsis model. These results further support the supposition that atl gene products are involved in cell separation, cell wall turnover and penicillin‐induced lysis of the cells.

Subcellular Localization of the Major Autolysin, ATL and Its Processed Proteins in Staphylococcus aureus

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138‐kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51‐kDa endo‐β‐N‐acetylglucosaminidase (51‐kDa GL) and 62‐kDa N‐acetylmuramyl‐L‐alanine amidase (62‐kDa AM). To investigate cell‐associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS‐stable cell‐associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138‐, 115‐, 85‐, 62‐ and 51‐kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138‐ and 115‐kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl:: Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138‐kDa protein is an ATL with a cleaved signal sequence; the 115‐and 85‐kDa proteins are intermediates; and the 51‐ and 62‐kDa proteins are cell‐associated 51‐kDa GL and 62‐kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138‐kDa ATL undergoes processing through intermediate proteins (115‐ and 85‐kDa proteins) to mature as the active cell cluster‐dispersing enzymes 51‐kDa GL and 62‐kDa AM on the cell surface.

Innate defences against methicillin-resistant Staphylococcus aureus (MRSA) infection

The innate immune system is the primary defence against bacterial infection. Among the factors involved in innate defence, anti‐microbial peptides produced by humans have recently attracted attention due to their relevance to some diseases and also to the development of new chemotherapeutic agents. Staphylococcus aureus is one of the major human pathogens, causing a variety of infections from suppurative disease to food poisoning. Methicillin‐resistant S. aureus (MRSA) is a clinical problem and with the recent emergence of a vancomycin‐resistant strain, this will pose serious problems in the near future. In investigating the molecular biology of S. aureus infections to develop new chemotherapeutic agents against MRSA infections, knowledge of the interaction of innate anti‐microbial peptides with S. aureus is important. In vitro and in vivo experiments demonstrate that exposure of S. aureus to host cells can induce the anti‐microbial peptides β‐defensin‐2 (hBD2), hBD3, and LL37/CAP18. The induction level of these peptides differs among strains, as does the susceptibility of the strains, with MRSA strains exhibiting lower susceptibility. In summary, the susceptibility of S. aureus strains, including MRSA strains, to components of the innate immune system varies, with the MRSA strains showing more resistance to both innate immune factors and chemotherapeutic agents. Copyright

Cell-wall thickness : possible mechanism of acriflavine resistance in meticillin-resistant Staphylococcus aureus

Acriflavine resistance in the clinical meticillin-resistant Staphylococcus aureus isolate KT24 was found not to be mediated by multidrug efflux pumps encoded by qacA/B, smr, qacE, qacG, qacH, qacJ or norA. Early uptake and accumulation of ethidium bromide in MRSA KT24 was significantly lower than that in a susceptible strain, although the efflux rates were similar. Therefore, a permeability barrier in MRSA KT24 may be the conceivable mechanism of acriflavine resistance. Interestingly, it was found that MRSA KT24 had a significantly thickened cell wall, and that cell-wall thickness increased gradually during bacterial growth. In contrast, cell size and surface area in MRSA KT24 were not different from those in the susceptible strain. Moreover, MRSA KT24 exposure to sub-MIC concentrations of acriflavine resulted in a thicker cell wall. These results indicate that cell-wall thickness may be responsible for acriflavine resistance in S. aureus.