Immunochemistry of antigens from aerobically and anaerobically grownStaphylococcus aureus

Immunochemical analyses of the major cell-wall antigens (teichoic acid, mucopeptide, and protein A) demonstrated quantitative but not qualitative differences between these antigens prepared from aerobically and anaerobically grownStaphylococcus aureus 7167. The reduced rate of oxygen uptake observed in anaerobically grownS. aureus 7167, indicating a repressed electron transport system and a reduction in the number of proteins (enzymes) associated with this system, was interpreted as evidence for the significant reduction in the number of protein antigens observed in immunochemical analyses of cell homogenate and intracellular antigen fractions prepared from anaerobically grown cells. Immunoelectrophoretic analyses indicated that a quantitatively greater amount of polysaccharide capsular material was obtained from anaerobically grownS. aureus 7167, and a relationship betweenin vivo pathogenicity and anaerobiosis was proposed.     

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents’ and children’s mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14 K lytic enzyme is derived from a microbial source.     

Chemical and ultrastructural studies on the cell wall ofStaphylococcus simulans biovarstaphylolyticus

Cell walls of strains ofStaphylococcus simulans biovarstaphylolyticus andS. aureus FDA 209P were compared ultrastructurally and chemically to investigate the mechanism of resistance of this strain ofS. simulans to its own staphylolytic endopeptidase. Chemical analysis of the peptidoglycans of the various strains examined showed that cells that were more resistant to lysis by the endopeptidase had lower glycine/serine ratios in their cross bridges and that, within a species, the more resistant cells had either fewer residues in these cross bridges or fewer cross bridges. Ultrastructural studies showed that cell wall thickness was not involved in resistance to the enzyme. Comparisons of the endopeptidase susceptibility of intact cells and isolated peptidoglycans from these cells suggested that the three-dimensional structure of the cell wall may play a role in resistance to lysis by the endopeptidase.     

Isolation and Characterization of Autolysin-Defective Mutants of Staphylococcus aureus That Form Cell Packets

Staphylococcus aureus produces multiple bacteriolytic enzymes (autolysins) and grows usually as a mixture of single cells, pairs, short chains, and irregular clusters. Autolysin-defective mutants that form cubic cell packets (Pa4A and PaH13) or grape-like clusters (Cu9S and CuD10) were isolated from S. aureus FDA 209P after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Pa4A mutant grown in nutrient broth formed cell packets consisting of 8–64 cells that appeared regularly arranged in three dimensions. Thin-section electron micrographs revealed that the packet cells were encased in an orderly manner within a thick peripheral wall and that their septa failed to split. Zymographic analysis of enzyme extracts from mutant Pa4A showed that it lacked the 33-kDa autolytic enzyme band present in the parent strain. Another mutant, Cu9S, formed grape-like clusters and showed a single autolytic enzyme band (33-kDa). The possibility that the 33-kDa autolytic enzyme is involved in splitting of the septum prior to cell separation in S. aureus is discussed. Received: 26 September 1996 / Accepted: 3 December 1996     

Electron Microscopy of the Lysis of Staphylococcus aureus Cell Walls by Aeromonas Lytic Factor

Isolated and purified cell walls of Staphylococcus aureus were treated with a purified fraction of the culture supernatant fluid of a species of Aeromonas. The course of lysis of the cell walls was followed over a period of time by examination of samples under an electron microscope. The undifferentiated cell wall was rapidly digested, but the equatorial rings were more resistant. The undifferentiated cell wall became a very thin sheet before completely dissolving, leaving a series of equatorial rings of various widths. As digestion proceeded, solubilization of the entire cell wall occurred. Analogous findings were obtained with purified S. aureus mucopeptide. It is concluded that the Aeromonas lytic principle is an enzyme, and that susceptible bonds are more concentrated in the undifferentiated cell wall mucopeptide.     

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.     

On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus

In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA^Gly isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA^Gly genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA^Gly isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA^Gly molecules (including the “pseudogene-encoded” tRNA^Gly) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the “pseudo”-tRNA^Gly(UCC) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA^Gly molecules to form ternary complexes with activated S. aureus EF-Tu·GTP. These tRNA^Gly molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA^Gly isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

Electron Microscopic Studies on the Outer Layers of Staphylococcus aureus Using a Lytic Enzyme from Flavobacterium

The structure of the outer layers (cell wall and membrane) of Staphylococcus aureus was studied by electron microscope using a bacteriolytic enzyme from Flavobacterium sp. called the L-11 enzyme. Comparative studies on the morphology of bacteria before and after treatment with this enzyme and cell wall and membrane fractions obtained from bacteria after the enzyme treatment led to the following conclusions. (1) The cell wall of S. aureus is composed of morphologically distinct two layers which are both susceptible to the L-11 enzyme. (2) Between the cell wall and membrane, there is an electron opaque region which could not be stained using any of the methods tested. (3) Before treatment of bacteria with the enzyme the cell membrane could not be seen clearly. However, after enzyme treatment the membrane was clearly seen. (4) The infolding of the inner layer of the cell wall, forming a structure like a mesosome, was liberated by extensive enzyme treatment.     

Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase

On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase.     

Surface-Bound Nuclease of Staphylococcus aureus: Localization of the Enzyme

The cellular localization of staphylococcus nuclease, previously known as an exoenzyme, was investigated, and the following results were obtained. (i) When Staphylococcus aureus cells were converted to protoplasts by cell wall lytic enzyme L-11 (a bacteriolytic enzyme purified from Flavobacterium sp. which specifically hydrolyzes amide and peptide linkages of murein layers), over 80% of the cell-bound nuclease was released into the surrounding sucrose medium. (ii) The cell-bound nuclease was associated with the cell-wall membrane fraction of mechanically disrupted cells. (iii) The nuclease activity of cell-wall membrane fractions from cells during early and late stages of protoplast formation were compared. Less activity was found in the late stage. These results suggest that nuclease may be located at or near the surface of the cells. The distribution of cell-bound nuclease in the cell-wall membrane fraction varied with the growth conditions of S. aureus. The activity of alkaline phosphatase, another surface enzyme, was also investigated. Less of this enzyme than nuclease was released when the cells were converted to protoplasts.     

Antibacterial activity of human mononuclear leukocytes againstStaphylococcus aureus

Human mononuclear leukocytes killStaphylococcus aureus cellsin vitro. The killing of the bacteria takes place even in the absence of antibodies. The presence of antibodies (in an autologous inactivated serum) usually enhances the antibacterial activity of mononuclear leukocytes. In some cases, however, this activity is markedly decreased by the serum, probably depending of the spectrum of antibodies contained in the serum. The antibacterial activity of mononuclear leukocytes is mostly due to monocytes because their depletion causes substantial drop or the activity disappearance. We failed to demonstrate in the case ofS. aureus the antibacterial cytotoxicity of T lymphocytes described by some authors dealing with Gram-negative bacteria. Large differences in the structure of the bacterial cell wall underlie apparently the different sensitivity of G⁺ and G⁻ bacteria to some protective mechanisms of the host. In the antibacterial assay againstS. aureus, electron microscopy revealed a maximal activation of monocytes which phagocytized the bacteria although extracellular killing is not excluded. Electronoptical findings point also to a possible participation of NK cells in the antibacterial cytotoxicity againstS. aureus.     

The Mechanism of Action of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Positive Bacteria: The Role of the Cell Wall Anionic Polymers of Target Bacteria

The study of the extracellular bacteriolytic enzymes of Lysobacter sp. showed that they can efficiently hydrolyze the peptidoglycan of gram-positive bacteria provided that there is an electrostatic interaction of these enzymes with the cell wall anionic polymers, teichoic and teichuronic acids in particular. The hydrolytic action of bacteriolytic enzymes on the cell wall largely depends on the negative charge of the teichoic and teichuronic acids rather than on their chemical composition.     

Surface-Bound Nuclease of Staphylococcus aureus: Purification and Properties of the Enzyme

The surface-bound nuclease of Staphylococcus aureus liberated during formation of protoplasts was purified 1,000-fold by chromatography on phosphocellulose. Its properties were compared with those of the known extracellular nuclease, purified 200-fold by the same procedures. The adsorbance of the surface-bound nuclease on phosphocellulose was distinctly different from that of the extracellular nuclease, but other properties of the two enzymes were similar. Both enzymes had a pH optimum of about 10 and required Ca²⁺ for activity. Both enzymes hydrolyzed deoxyribonucleic acid (DNA) and ribonucleic acid, and denatured DNA was a better substrate than native DNA. Both enzymes were inhibited by the same metal ions. Nuclease-less mutants of S. aureus were isolated from S. aureus 209P by using N-methyl-N′-nitroso-N-nitrosoguanidine. These mutants contained neither surface-bound nor extracellular nuclease activity. These results suggest that the surface-bound and extracellular nucleases are expressed from the same cistron of S. aureus.     

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.     

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

Reduction of the Peptidoglycan Crosslinking Causes a Decrease in Stiffness of the Staphylococcus aureus Cell Envelope

We have used atomic-force microscopy (AFM) to probe the effect of peptidoglycan crosslinking reduction on the elasticity of the Staphylococcus aureus cell wall, which is of particular interest as a target for antimicrobial chemotherapy. Penicillin-binding protein 4 (PBP4) is a nonessential transpeptidase, required for the high levels of peptidoglycan crosslinking characteristic of S. aureus. Importantly, this protein is essential for β-lactam resistance in community-acquired, methicillin-resistant S. aureus (MRSA) strains but not in hospital-acquired MRSA strains. Using AFM in a new mode for recording force/distance curves, we observed that the absence of PBP4, and the concomitant reduction of the peptidoglycan crosslinking, resulted in a reduction in stiffness of the S. aureus cell wall. Importantly, the reduction in cell wall stiffness in the absence of PBP4 was observed both in community-acquired and hospital-acquired MRSA strains, indicating that high levels of peptidoglycan crosslinking modulate the overall structure and mechanical properties of the S. aureus cell envelope in both types of clinically relevant strains. Additionally, we were able to show that the applied method enables the separation of cell wall properties and turgor pressure.     

Identification of novel bacterial histidine biosynthesis inhibitors using docking,ensemble rescoring,and whole-cell assays

The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring, that is sufficiently accurate to justify immediate testing of the identified compounds by whole-cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of ～10⁶ compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus- and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway.