Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner.

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

Induction of necrosis factor-alpha and interleukin-6 in mice in vivo and in murine peritoneal macrophages and human whole blood cells in vitro by Micrococcus luteus teichuronic acids.

Earlier studies showed that Micrococcus luteus cells and cell walls induced anaphylactoid reactions leading to death, in some instances within 1 h, in C3H/HeN mice primed with muramyl dipeptide (MDP). They also induced serum cytokines in the surviving mice. The present study investigated the structural components responsible for these activities. Teichuronic acids, a component of M. luteus cell walls, induced tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in MDP-primed C3H/HeN mice. Peptidoglycans had little effect on the cytokine-inducing activities. Reducing teichuronic acids, i.e., teichuronic acids whose carboxyl groups had been reduced, lost their cytokine-inducing activities. Neither peptidoglycans nor teichuronic acids induced anaphylactoid reactions in the MDP-primed mice. Purified teichuronic acids also induced TNF-alpha and IL-6 production in C3H/HeN murine peritoneal macrophages and human whole-blood cells in the culture, but reduced teichuronic acids did not. The purified teichuronic acids induced no TNF-alpha and only low levels of IL-6 in MDP-primed C3H/HeJ mice, and neither cytokine in peritoneal macrophage cultures from C3H/HeJ mice with a single point of mutation in Toll-like receptor 4 (TLR4) gene. These findings suggest that induction of cytokines by teichuronic acids is mainly TLR4-dependent.

Micrococcus luteus cells and cell walls induce anaphylactoid reactions accompanied by early death and serum cytokines in mice primed with muramyl dipeptide.

Micrococcus luteus strains at a dose of 500 microg of whole cells caused anaphylactoid reactions leading to death in some instances within 1 h in C3H/HeN mice primed with muramyl dipeptide (MDP, 100 microg). Tumor necrosis factor (TNF) and interleukin-6 (IL-6) were induced in the serum of half and of all the surviving mice, respectively. Cell wall specimens of M. luteus so far examined also caused anaphylactoid reactions accompanied by early death and one strain induced high levels of TNF and IL-6. Cytoplasmic membranes also induced IL-6. Essentially similar results were obtained with representative M. luteus cells and a cell wall specimen in MDP-primed C3H/HeJ mice. These results indicate that M. luteus has virulence activities that are associated with the induction of septic shock and systemic inflammatory diseases.

Water molecules bound to fibrils by hydrogen bonds play an important role on the surface fibrillar structure of Candida albicans cells.

Influence of surrounding media on the appearance of surface structure of Candida albicans was further investigated by rapid-freezing and freeze-fracturing techniques with a scanning electron microscope. Fibrillar structure was observed on the surface of the cells treated with water, chloroform, trichloroethane, toluene or isoamyl alcohol, but not on that of the cells treated with methanol, ethanol, isopropanol, n-butanol, acetic acid or acetone. The appearance of the fibrillar structure is proposed to be discussed in a viewpoint of the chemical interactions among the constituent molecules of a fibril, water molecules bound to the fibril molecules and the molecules of surrounding medium, especially a role of water molecules bound to the fibril molecules by hydrogen bonds.

Cell separation system studied by mixed culture of single wild strain with tetrads-forming mutant strain of Micrococcus luteus.

Mixed culture study of singly occurring wild strain IFO 3333 of Micrococcus luteus and a tetrads-forming mutant strain MT, in the absence or presence of trypsin, supported our previous assumption that at least two kinds of separation systems were involved in cell separation of M. luteus, the one having a physiological role in cutting off the outermost layer of the cell wall (separation system-Om) and the other in cutting off the inner layer of the "proper" cell wall or the septum (separation system-In). The separation system-Om of IFO 3333 insensitive to trypsin substituted, freely from the cells, for that of MT sensitive to trypsin.

Cell surface of a tetrads-forming mutant of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface.

Tetrads-forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four "unit monococci" constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild-type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165-174, 1989) and the mutant MT cells is discussed.

Chemical treatments of the cell packets induced from a Micrococcus luteus mutant and teichuronic acids on the packet surface.

Cell packets (MT packets) induced from a tetrads-forming mutant (strain MT) of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of MT packets with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the MT packets, mediated with the antiserum, was also observed with SEM. Gold particles were observed uniformly on the whole packet surface and also on the bridging structure formed by the outermost layer of the cell wall. Mild acid treatment, NaIO4-NaBH4 treatment and mild Smith degradation of the MT packets extremely decreased the agglutinability and binding of protein A-gold particles. The treatments gave a little influence on the surface feature and appreciably destroyed the regular packet structure. It was supposed that teichuronic acids distributed uniformly on the whole packet surface, naturally on the surface of the bridging structure too, and appreciably participated in the maintenance of the regular packet structure.

Comparison of two kinds of cell packets of Micrococcus luteus by scanning electron microscopy: outermost layer maintaining the packet structure.

Two kinds of cell packets of Micrococcus luteus, one having teichuronic acids (TUA) in the cell wall and the other lacking TUA, have been independently reported by two groups of workers. A comparison by scanning electron microscopy of these packets provided a possibly consistent interpretation for the seemingly conflicting opinions whether TUA were involved in packet induction. It was strongly suggested that the packets having TUA in the wall were rigidly maintained by a bridging structure of the outermost layer of the peripheral wall, while the packets lacking TUA showed low contribution of the outermost layer to the bridging structure probably due to the absence of TUA.

Influence of surrounding media on preservation of cell wall ultrastructure of Candida albicans revealed by low temperature scanning electron microscopy.

Influence of surrounding media on the surface structures of the cell wall of Candida albicans was discussed with respect to the preservation of ultrastructure during the specimen preparation for scanning electron microscopy. The fibrillar structure of the cell surface was distinctly identified by the rapid-freezing technique. It was difficult, however, to observe this structure by the conventional specimen preparation technique. The reason for the difference between these two preparation techniques was studied using a low temperature SEM. Through investigating the influence of each step of the conventional technique on the fibrillar structure, it was found that the fibrils were drastically deformed and disappeared during the dehydration step in ethanol above 80% in concentration. In order to study which physicochemical properties participated in this disappearance phenomenon, yeast cells were treated with various media: solutions in different pH ranges and at different salt concentrations, ionic solutions, surfactants, formamide, dimethyl sulfoxide, acetone and Fehling's solution. As a result, the fibrillar structure was found well preserved when the medium had an affinity for the constituent molecules of the fibrils. When without affinity, the fibrils suffered a remarkable deformation. The mechanism of this deformation is discussed in terms of molecular interaction of solute and solvent.

Cell surface of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface.

Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders--the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.

Cell wall-bridge maintaining three dimensional structure of cell packets formed by the localized suppression of cell separation of a Micrococcus lysodeikticus (luteus) mutant.

Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells. The mechanism of cell wall-bridge formation is discussed in connection with cell separation.

Demonstration of the physiological role of autolysis by a comparative study with a wild-type and its non-autolytic mutant of Micrococcus lysodeikticus (luteus) cultivated with externally added proteolytic enzymes.

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.

Process of consecutive cell divisions and separations in a regular tetrads-forming mutant of Micrococcus lysodeikticus (luteus).

A mutant MT of Micrococcus lysodeikticus (luteus) IFO 3333, whose minimum growing unit is not a single cell, but a tetrad unlike the wild-type divides by binary fission of each monococcus, and then separates first into two daughter tetrads, second into four tetrads and third into eight tetrads. The three planes of either the cell division or the cell separation are equivalent to one another and oriented at right angles in three dimensions, respectively. The process of consecutive cell divisions and separations of the mutant tetrads was schematically illustrated.