Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

The study of Staphylococcus aureus strain resistant to actinomycin D]

Staphylococcus aureus strains, resistant to actinomycin D (AMD) and to gramicidin S (GS) were selected by S. aureus 209P passing on the media containing the above mentioned drugs. Strain R80 resistant to AMD and strain R9 resistant to GS and AMD and described before didn't perform enzyme inactivation of AMD. Cells of both strains had diminished ability to bind exogenous AMD. Electron microscopy investigation revealed that cells of R80 strain had thickened cell walls and they are characterized by more electron density then cells of R9 strain and of parent strain. Adaption to AMD and GS influenced also on functions of some staphylococcal surface proteins--the activity of endogenous coagulase (clumping factor) was found only in R9 strain. Exogenous coagulase was present in all the strains, but development of resistant to AMD and GS diminished this enzyme activity. It is concluded that development of resistance to AMD and GS causes substantial changes in staphylococcal cell wall, but the type of these changes differ.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

Study of the adaptation resistance in bacteria to membrane active polypeptide antibiotics

Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin. The sensitive and resistant cells did not differ by their ability to bind gramicidin. Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant. Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M. lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations. Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin. It is likely that during adaptation of M. lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Investigation of cell wall components in actinomycin D resistant Staphylococcus aureus]

Cell walls in 2 strains of Staphylococcus aureus 209P, i.e. actinomycin D susceptible and resistant ones were comparatively investigated. The resistant cells contained much more wall material per a unit of the biomass weight vs the susceptible strain cells, that conformed to thickening of the resistant cell walls detected by electron microscopy and a sharp increase of their electron density. Investigation of peptidoglycans and teichoic acids did not reveal any significant alterations in the structure of the wall components in the actinomycin D resistant cells. Only some increase of glucosamine in the peptidoglycan fraction of the resistant cells vs the susceptible ones was observed. It was shown that preparations of the resistant cell walls and peptidoglycan isolated from the resistant cells were able to bind somewhat lower quantities of actinomycin D vs the analogous preparations of the susceptible cells. The significant decrease of the antibiotic binding by live cells of the resistant strain probably slightly depended on the structure characteristics of the main wall components. The barrier properties of the walls in resistant staphylococci are most likely defined by the wall thickening and consolidation while adapting to actinomycin D.     

Quantitative determination of the protein and carbohydrate polymers in the cell wall of Streptococcus group A

The content of protein and carbohydrate polymers was estimated in the cell wall of Streptococcus, group A, type 29. A method was developed for analysing peptidoglycane in a polysaccharide-peptidoglycane complex after the prior oxidation by sodium periodate. It was found that the cell wall peptidoglycane bears two carbohydrate and three amino acid residues, i. e. N-acetylglucosamin, muramic acid, glutamic acid, alanine and lysine, in the ratio 1:1:1:4:1, respectively. The data on the cell wall composition prior to and after its oxidation with sodium periodate are given, and the ratio between the main structural components is determined: proteins (60% mol), polysaccharide (23% mol), peptidoglycane (17% mol).     

Lysis of Staphylococcus aureus Cell Walls by a Soluble Staphylococcal Enzyme

Enzyme preparations of Staphylococcus aureus were examined for their ability to solubilize (32)P-labeled cell walls of the parent organism. Enzymatic activity was observed in the growth medium, in soluble fractions, and associated with native cell walls. Enzyme associated with isolated cell walls could be inactivated with formaldehyde without reducing the susceptibility of the walls to the action of added enzyme. When cells are frozen and thawed, 50 to 75% of the intracellular enzyme is released along with 2% of the intracellular protein. This freeze-thaw extracted enzyme has little, if any, activity on intact S. aureus cells. It appears that the enzyme resides near the cell wall and acts on the cell-wall inner surface.     

Isolation and characterization of a mutant of Staphylococcus aureus deficient in autolytic activity.

A mutant of Staphylococcus aureus H (RUS3) uas isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rate of autolysis of whole cells and isolated cell walls of RUS3 was less than 10% of the parent strain. In addition, the ability of the crude soluble enzyme isolated from RUS3 to degrade cell walls was negligible compared with the parent strain. The cell wall composition and the generation time of RUS3 were comparable to the parent strain. Unlike S. aureus H, RUS3 grew in clumps and did not undergo cell wall turnover. Both strains exhibited identical kinetics of killing by penicillin G. This may indicate that autolytic enzymes play a role in cell wall turnover and cell separation, but in S. aureus most of the autolytic activity is unrelated to the lethal effect of cell wall antibiotics.     

Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 microg/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 microg/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.     

Autolysis in Staphylococcus aureus: Preferential Release of Old Cell Walls

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.     

Stability of Escherichia coli to the membranotropic antibiotic gramicidin S]

The work was aimed at studying the effect of gramicidin S on the intact cells, spheroplasts and membrane specimens of Escherichia coli K12S with the natural resistance to this antibiotic. The resistance was shown to be caused by the barrier properties of the cell wall: the spheroplasts were highly sensitive to the lytic action of gramicidin S. The differences in the sensitivity to gramicidin S of substrate oxidation carried by the membranes of E. coli and Micrococcus luteus, a sensitive organism, were not of crucial significance for the manifestation of the resistance. The resistance was not associated with the decrease of gramicidin S adsorption: the cells were capable of binding large quantities of the antibiotic and remaining viable. Gramicidin S appeared to be attached to the cell walls (most likely, the outer membranes) rather than the cytoplasmic membranes.     

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608-619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(-X-D-Leu-D-Lys-D-Leu-L-Pro-)2, where X = Gly, D-Leu and D-cyclohexylalanine (D-cHxAla); (2) N,N'-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a beta-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = D-cHxAla induced the efflux of K+ through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K+ from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Effect of salvin on the synthesis of macromolecules in Staphylococcus aureus 209P

The effect of salvin and its active component, carnazolic acid, on the synthesis of macromolecular compounds in the cells of S. aureus 209P was studied. It was shown that the inhibitory action of salvin on the synthesis of peptidoglycane in the culture was defined by the presence of carnazolic acid in its composition. In the bactericidal concentration, carnazolic acid was twice as less active as salvin and inhibited incorporation of labeled precursors into RNA and protein. The findings grounded the conclusion that some nonidentified components of salvin with low antimicrobial activity contained in it in insignificant quantities had an additional inhibitory effect on the process. Comparative study of salvin and antibiotics with the known mechanisms of action such as benzyl penicillin or chloramphenicol revealed a certain similarity in the action of salvin and benzyl penicillin on incorporation of labeled precursors into the macromolecular compounds of S. aureus 209P.     

Organization and chemical composition of the cell wall of gramicidin S-producing Bacillus brevis

The morphology of cells and cell walls was studied in the Bacillus brevis G.-B. R form during its growth and gramicidin S accumulation in it. The membrane apparatus became more complicated and certain other morphological changes were detected in the cells with aging. The cell wall was rather complex even in young cells and consisted of three electron-dense layers where the external and internal layers had an ordered structure. Only the external layer underwent some modifications in the course of growth and these coincided in time with the beginning of intensive gramicidine S biosynthesis. However, the three-layer structure of the cell wall and the ordered organization of the external and internal layers remained unchanged. A preparation of cell walls and preparations of their external and internal layers were isolated from cells synthesizing gramicidine S in the amount of 20 micrograms/ml of the cultural broth. An acid protein having the molecular mass of 100 kD was shown to be the major component of the external layer according to the data of electrophoresis in PAAG with SDS. The middle layer was sensitive to lysozyme, did not have a ordered structure on electron micrographs, and consisted mainly of peptidoglycan.     

Mutual relationship between antibiotics and resting spores of Bacillus subtilis: morphological changes and macromolecular synthesis after germination of spores treated with cyclic polypeptide and aminoglycoside antibiotics

Morphological changes and synthesis of DNA, RNA, protein, and cell wall were investigated during germination of resting spores of Bacillus subtilis exposed transiently to the cyclic polypeptide antibiotics, polymyxin B and gramicidin S, and the aminoglycoside antibiotics, streptomycin, kanamycin, and gentamicin. Normal germinated spores showed breaks of the spore coat, a diminution in size and a fibrillar appearance of the cortex, a swelling core, a cell wall as thick as that of vegetable cells, some mesosomes and DNA fibrils. On the other hand, no breaks of the spore coat, a spore core with a slight swelling and irregular form, a thin cell wall, no demonstration of the nuclear material and no granularity in the cytoplasm were characteristic of the germinated spores derived from polymyxin B- and gramicidin S-treated resting spores. With gramicidin S-treated germinated spores a few vacuoles were formed in the cytoplasm. Both polymyxin B- and gramicidin S-treated germinated spores showed little or no synthesis of DNA, RNA, and protein. The vegetative cells derived from streptomycin-treated resting spores demonstrated several finely granular regions in the cytoplasm and a disorder of the fibrillar nucleoid, and their autolysis occurred early. Their DNA and RNA synthesis was normal, whereas protein synthesis was low. In spite of no occurrence of cell division and very low protein synthesis, the most striking characteristics of the outgrowing cells derived from kanamycin-treated resting spores were a markedly thickened cell wall and a continuous incorporation of labeled D-alanine suggesting cell wall synthesis; RNA synthesis was slightly lower and DNA synthesis was almost normal. The outgrowing cells from gentamicin-treated resting spores also revealed relatively thick cell walls and a very slight incorporation of labeled D-alanine. Their DNA and RNA synthesis was fairly low and protein synthesis was almost completely inhibited. These results coincide with the growth curves of individual antibiotic-treated resting spores.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.