Modification of autolysis by synthetic peptides derived from the presumptive binding domain of Staphylococcus aureus autolysin.

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10- to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of A11, may be important segments for Atl to bind to the cell surface.

Tn551-mediated insertional inactivation of the fmtB gene encoding a cell wall-associated protein abolishes methicillin resistance in Staphylococcus aureus.

A Tn551 insert in a gene termed fmtB was shown to reduce oxacillin as well as Triton X-100 resistance in highly methicillin-resistant Staphylococcus aureus (MRSA) COL. Backcrosses of fmtB::Tn551 into S. aureus COL and into two genetically distinct MRSA strains, KSA8 and NCTC10443, confirmed the linkage of fmtB::Tn551 with loss of oxacillin resistance. The fmtB gene codes for a protein of a deduced molecular mass of 263 kDa that contains 17 tandem repeats of 75 amino acids and a C-terminal LPXTG cell wall-sorting motif. Immunoblots with anti-FmtB antibodies confirmed its localization in the cell wall fraction. The fmtB gene was mapped downstream of the phosphoglucosamine mutase operon glmM which catalyses formation of glucosamine-1-phosphate. Oxacillin resistance was not restored in fmtB mutants by trans-complementation with fmtB. However, although GlmM production was not affected by fmtB inactivation, oxacillin resistance was increased in fmtB mutants by introducing a plasmid-borne glmM gene, presumably by GlmM overexpression. Interestingly, a similar phenotypic complementation was obtained in fmtB mutants by including substrate level concentrations of N-acetylglucosamine or glucosamine in the growth medium. Inactivation of the fmtB gene seems therefore to have an indirect effect on methicillin resistance which can be relieved by increasing the production of the cell wall precursor glucosamine-1-phosphate.

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus.

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.

Triton X-100-induced lipoteichoic acid release is correlated with the methicillin resistance in Staphylococcus aureus.

We previously reported that Triton X-100 (TRX) reduced methicillin resistance in Staphylococcus aureus, although the degree of reduction varied among strains. One of the biological effects of TRX on S. aureus cells was enhancement of lipoteichoic acid (LTA) release. We investigated the correlation between the amount of LTA released and the degree of reduction in methicillin resistance induced by TRX. The strains showing the greatest reduction of methicillin resistance released the largest amount of LTA, compared to those showing slight or moderate reduction. A mutant whose resistance was not affected by TRX did not increase its release of LTA. These findings suggest that LTA release is associated with a reduction in methicillin resistance in the presence of TRX.

Effect of clarithromycin on Pseudomonas aeruginosa biofilms.

Using an experimental in vitro culture system, we investigated the effect of clarithromycin on biofilm formation by a leucine-requiring Pseudomonas aeruginosa mutant strain (HU1). Biofilm formation on celldesks in a chemically defined medium was assessed by viable cell count as well as by measurement of glycocalyx production and scanning electron-microscopic observation. Cells proliferated exponentially until day 3 and remained stationary afterwards. The amount of glycocalyx, simultaneously semiquantified, showed a linear increase from day 1 to day 12. Scanning electron microscopy revealed firm biofilms on day 5. Three different concentrations of Clarithromycin (CAM) (minimum inhibitory concentration MIC 64 microg/ml) were added continuously at the early and late phases of biofilm formation, and the antibiofilm effect of CAM was evaluated by the changes in cell count and glycocalyx production. CAM was effective on biofilms at 100 microg/ml but neither at 1 nor at 10 microg/ml. It is suggested that glycocalyx production started following bacterial multiplication and continued even after the cells had entered the stationary phase to form mature biofilms. No antibiofilm effect of CAM was observed at sub-MIC.

Cytotoxic necrotizing factor type 2 produced by pathogenic Escherichia coli deamidates a gln residue in the conserved G-3 domain of the rho family and preferentially inhibits the GTPase activity of RhoA and rac1.

Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122(RhoGAP). Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.

Expression of osteoprotegerin (osteoclastogenesis inhibitory factor) in cultures of human dental mesenchymal cells and epithelial cells.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) inhibits osteoclast differentiation, activity, and survival; therefore OPG/OCIF may regulate the resorption of dental hard tissues, such as alveolar bone, cementum, and dentin. To investigate this issue, reverse transcriptase-polymerase chain reaction using specific primers for OPG/OCIF was performed with total RNAs isolated from human gingival keratinocytes (HGKs), human gingival fibroblasts (HGFs), human periodontal ligament cells (HPDLs), and human pulp cells (HPCs) in culture. PCR products were found in HGFs, HPDLs, and HPCs, but not in HGKs, and the DNA sequence of these products was 100% identical to the reported sequence of the OPG gene. Northern blot analyses also showed that HGFs, HPDLs, and HPCs, but not HGKs, expressed OPG/OCIF transcripts of approximately 2.5 kb. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased OPG/OCIF mRNA levels in a dose-and time-dependent manner in HPDL. After 12 h of treatment, IL-1beta at 3 ng/ml and TNF-alpha at 3 ng/ml increased OPG/OCIF mRNA expression by 190% and 110%, respectively, with a maximal effect. The stimulatory effects of IL-1beta and TNF-alpha were also seen in HPC. However, IL-6 and transforming growth factor-beta had little effect on OPG/OCIF mRNA levels in HPDL. These findings suggest that OPG/OCIF synthesized by dental mesenchymal cells locally regulates the resorption of dental hard tissues through cytokines.

Characterization of fmtA, a gene that modulates the expression of methicillin resistance in Staphylococcus aureus.

FmtA is a factor which affects the methicillin resistance level in methicillin-resistant Staphylococcus aureus. Since FmtA has two of three conserved motifs which are typically found in penicillin-binding proteins (PBPs) and beta-lactamases, we investigated the penicillin-binding activity of recombinant FmtA and found no such activity. Immunoblotting analysis revealed that FmtA localizes in the membrane fraction. To investigate the function of FmtA, high-pressure liquid chromatography analysis of cell wall muropeptides was performed with an fmtA-inactivated mutant and its parent. The mutant showed a reduced cross-linking and partially reduced amidation of glutamate residues in the peptidoglycan of the mutant. The transcription of fmtA was dose dependently increased by the addition of beta-lactam antibiotics, fosfomycin, and bacitracin, while its transcription was not changed by the addition of vancomycin or tetracycline. These results reveal that Fmt is a membrane-located, non-penicillin-binding protein and that mutation of fmtA affects the cell wall structure, although its precise function is still unknown.

Cloning and characterization of a gene, pbpF, encoding a new penicillin-binding protein, PBP2B, in Staphylococcus aureus.

A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and beta-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents: beta-lactams and fluoroquinolones.

The growth rate of biofilm bacteria of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in a chemically-defined medium. The semiquantitative measurement of glycocalyx and scanning electron microscopy revealed that the kinetics of HU1-biofilm formation were dependent on the incubation time and the leucine concentration in the medium. The effect of the growth rate of biofilm cells on their susceptibility to antimicrobial agents, three beta-lactams and four fluoroquinolones, was evaluated. beta-Lactams showed weak bactericidal activity to biofilm cells; the activity was greater in younger biofilm cells growing in high concentrations of leucine. Fluoroquinolones revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate. The following is suggested: the bactericidal action of beta-lactams against biofilm cells is affected by the cell growth rate, while that of fluoroquinolones is considerably greater and independent on the growth rate.

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein.

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.

Expression analysis of the autolysin gene (atl) of Staphylococcus aureus.

The bifunctional autolysin gene (atl) of Staphylococcus aureus was transcribed into a 4.1-kb transcript. The transcription initiation site was located at an adenine residue 33-nt upstream from the putative atl start codon. Analysis using a promoter-reporter plasmid showed that promoter activity increased during the exponential growth phase. The Tn551 insertion site of the autolysis-deficient mutant S. aureus RUSAL2 was located in the putative catalytic region of the glucosaminidase domain.

The cell cycle-specific growth-inhibitory factor produced by Actinobacillus actinomycetemcomitans is a cytolethal distending toxin.

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi. The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G2/M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

The mechanism of sensitizing effect of a triazine dye, cibacron blue F3GA, on methicillin-resistant Staphylococcus aureus to oxacillin.

We recently found that a triazinyl dye, cibacron blue F3GA (CB), has a sensitizing effect on the in-vitro susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin (C. Shirai, M. Sugai, H. Komatsuzawa, K. Ohta, M. Yamakido, H. Suginaka, Antimicrob. Agents Chemother. 42: 1278-1280, 1998). Among nine triazinyl dyes tested, CB the strongest sensitizing effect. Population analysis demonstrated that CB reduced the resistance level of MRSA. In the presence of oxacillin at subinhibitory concentrations, CB inhibited the growth of MRSA, but its effect on the cells appeared to be bacteriostatic. Under experimental conditions, CB did not affect the amount of PBP2', binding of [14C]benzylpenicillin to PBP2', or peptidoglycan susceptibilities to bacteriolytic enzymes. Autolytic enzyme-deficient MRSA mutants were equally as sensitive as the parent strain to the effect of CB on the susceptibility to oxacillin. CB affected the resistance level of MRSA irrespective of the status of the mecI/mecR1 element and/or penicillinase plasmids. The sensitivities of several bacteriolytic enzymes to heat-inactivated MRSA cells were not affected when the cells grown in the presence of CB. CB did not stimulate the release of lipoteichoic acid from the cells. These results suggest that the sensitization effect of CB cannot be simply explained by its effect on mecA related products, autolysins, femAB products or the release of lipoteichoic acid.

A triazine dye, cibacron blue F3GA, decreases oxacillin resistance levels in methicillin-resistant Staphylococcus aureus.

Cibacron blue F3GA (CB) was found to reduce the MIC of oxacillin for methicillin-resistant Staphylococcus aureus (MRSA). This effect was not observed with methicillin-susceptible S. aureus. CB alters the resistance level of MRSA through a factor(s) other than mecA-related products, major autolysins, or femAB products. The exact target(s) of CB in causing the effect is unknown.

Effect of adherence on antimicrobial susceptibility of Pseudomonas aeruginosa, Serratia marcescens, and Proteus mirabilis.

A simple method was used for testing the antibiotic susceptibility of adherent bacteria to plastic surfaces. Pseudomonas aeruginosa, Serratia marcescens, and Proteus mirabilis cells adhering to the bottom of a plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After 24-h incubation the solutions were removed and a fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-h incubation. In our assay system, we employed a short incubation time (1-h) involving adherence of bacteria to a surface for the purpose of minimizing the effect of the glycocalyx on antibiotic activity. Even if the bacteria did not form a biofilm, the minimal bactericidal concentrations for adherent bacteria (MBCADs) markedly elevated. The MBCADs of ofloxacin well correlated with the bacteriological eradication by ofloxacin treatment for urinary tract infections (UTIs) associated with indwelling urinary catheters, whereas the minimal inhibitory concentrations did not show a correlation. Kinetic studies showed that adherent Pseudomonas aeruginosa had a 2 h-lag time before logarithmically growing when these bacteria were incubated in Mueller-Hinton broth without antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the antimicrobial chemotherapy of biofilm infections. Moreover, our assay system was considered to be useful in the therapeutic selection of antibiotics for these infections.

Zymographic characterization of Staphylococcus aureus cell wall.

Susceptibilities of several preparations of Staphylococcus aureus cells to various peptidoglycan hydrolases with known bond specificity were analyzed by zymography. The substrates were intact S. aureus cells, cells boiled in the presence of SDS and cells treated with trichloroacetic acid after treatment with boiling SDS solution (TCA-cells). Twofold dilutions of lysostaphin (LS), lysozyme (LZ), S. aureus 51 kDa glucosaminidase (GL) or S. aureus 62 kDa amidase (AM) were electrophoresed, and the minimal enzyme dose showing a visible bacteriolytic band was defined as MBD (minimal bacteriolytic dose). Under the same experimental conditions, this method gave reproducible results. As the substrate for zymogram, TCA-cells were the most sensitive to LS, LZ and AM, whereas the three substrate were equally sensitive to GL. A zymographic analysis of methicillin-resistant S. aureus treated with methicillin together with previous studies suggest that this method can be used for the preliminary characterization of S. aureus cell wall peptidoglycan.

Expression and purification of epidermal cell differentiation inhibitor (EDIN) from Bacillus subtilis.

The expression of staphylococcal epidermal cell differentiation inhibitor (EDIN), an ADP-ribosyltransferase targeting the small GTP-binding protein rho p21, was examined using Bacillus subtilis. A recombinant plasmid, containing B. licheniformis alpha-amylase promoter flanking either a beta-glucanase or a B. cereus sphingomyelinase signal sequence, and a DNA fragment corresponding to mature EDIN were constructed and used to transform B. subtilis KN2. Transformants were designated ED7 and ED8, respectively. ED7 extracellularly produced recombinant protein, which was purified to homogeneity through column chromatography using SP-Toyopearl 650 cation-exchange gel and the HA1000 hydroxyapatite HPLC column. ED8 did not grow in broth culture. Biochemical and biological studies of purified protein revealed that ED7 produced a correctly processed recombinant EDIN, indistinguishable from natural EDIN.

Cloning and characterization of the fmt gene which affects the methicillin resistance level and autolysis in the presence of triton X-100 in methicillin-resistant Staphylococcus aureus.

In methicillin-resistant Staphylococcus aureus (MRSA) strains, Triton X-100 reduced the oxacillin resistance level, although the degree of reduction varied from strain to strain. To study the responses of MRSA strains to Triton X-100, we isolated a Tn551 insertion mutant of the COL strain that became more susceptible to oxacillin in the presence of 0.02% Triton X-100. The Tn551 insertion of the mutant was transduced back to the parent strain, other MRSA strains (strains KSA8 and NCTC 10443), and methicillin-susceptible strain RN450. All transductants of MRSA strains had reduced levels of resistance to oxacillin in the presence of 0.02% Triton X-100, while those of RN450 did not. Tn551 mutants of KSA8 and NCTC 10443 also had reduced levels of resistance in the absence of 0.02% Triton X-100. The autolysis rates of the transductants in the presence of 0.02% Triton X-100 were significantly increased. Amino acid analysis of peptidoglycan and testing of heat-inactivated cells for their susceptibilities to several bacteriolytic enzymes showed that there were no significant differences between the parents and the respective Tn551 mutants. The Tn551 insertion site mapped at a location different from the previously identified fem and llm sites. Cloning and sequencing showed that Tn551 had inserted at the C-terminal region of a novel gene designated fmt. The putative Fmt protein showed a hydropathy pattern similar to that of S. aureus penicillin-binding proteins and contained two of the three conserved motifs shared by penicillin-binding proteins and beta-lactamases, suggesting that fmt may be involved in cell wall synthesis.

Permeation of antimicrobial agents through Pseudomonas aeruginosa biofilms: a simple method.

In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.