Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate- S. aureus-type resistance to vancomycin

Custom-designed gene chips (Affymetrix) were used to determine genetic relatedness and gene expression profiles in Staphylococcus aureus isolates with increasing MICs of vancomycin that were recovered over a period of several weeks from the blood and heart valve of a patient undergoing extensive vancomycin therapy. The isolates were found to be isogenic as determined by the GeneChip based genotyping approach and thus represented a unique opportunity to study changes in gene expression that may contribute to the vancomycin resistance phenotype. No differences in gene expression were detected between the parent strain, JH1, and JH15, isolated from the nares of a patient contact. Few expression changes were observed between blood and heart valve isolates with identical vancomycin MICs. A large number of genes had altered expression in the late stage JH9 isolate (MIC = 8 microg/ml) compared to JH1 (MIC = 1 microg/ml). Most genes with altered expression were involved in housekeeping functions or cell wall biosynthesis and regulation. The sortase-encoding genes, srtA and srtB, as well as several surface protein-encoding genes were downregulated in JH9. Two hypothetical protein-encoding genes, SAS016 and SA2343, were dramatically overexpressed in JH9. Interestingly, 27 of the genes with altered expression in JH9 grown in drug-free medium were found to be also overexpressed when the parental strain JH1 was briefly exposed to inhibitory concentrations of vancomycin, and more than half (17 of 27) of the genes with altered expression belonged to determinants that were proposed to form part of a general cell wall stress stimulon (S. Utaida et al., Microbiology 149:2719-2732, 2003).     

Glycine dependence of Staphylococcus aureus strains]

Glycine dependent Staphylococcus aureus var. bovis strains (TSCHAPE and RISCHE 1971) were tested for their ability to grow on glycine containing and glycine deprived media. We observed glycine dependence only in minimal media. In complete media the strains grew in absence of glycine. Testing the ability of some glycine precursors we found that in minimal media glycine could be replaced by threonine and in some cases by serine. One mutant (Gly 100 Glyox.r.) was able to metabolize glyoxylate instead of glycine. Aspartic acid was metabolized in presence of glycine. Using 14C-aspartic acid we detected 14C-glycine and 14C-threonine. Presumably the bacteria metabolize aspartic acid to glycine via threonine.     

Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus

Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.     

Salt-induced cell lysis of Staphylococcus aureus

Cell lysis was efficiently induced in Staphylococcus aureus by the addition of 0.3 m NaCl to exponentially growing cultures at 30°C. When cells harvested at the exponential phase were incubated in buffer with NaCl, autolysis occurred. Treatment with chloramphenicol failed to induce cell lysis by NaCl. The effects of NaCl on growing cells and harvested cells were inhibited by the addition of sodium polyanethole sulfonate, subtilisin, cardiolipin, and lipoteichoic acid. These agents diminished the activity of a cell wall-lytic enzyme liberated from the cells in the presence of NaCl. Lysis induced by salt appears to be catalyzed by a similar lytic enzyme in growing and harvested cells.     

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O(2)(-)), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O(2)(-), and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O(2)(-) increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O(2)(-) in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O(2)(-) causing more oxidative stress than in resistant S. aureus.     

Geometry of cell division in Staphylococcus aureus.

The process of division in Staphylococcus aureus was examined by phase-contrast microscopy. The organisms appeared to divide in three alternating perpendicular planes, with sister cells remaining attached to each other after division. The resulting point of attachment was usually not exactly at the point corresponding to the center of the previous septal disk. Moreover, sister cells often changed position with respect to one another while still remaining attached. These factors are apparently responsible for the irregularity of staphylococcal clumps. Studies with penicillin and the examination of thin sections in the electron microscope confirm the conclusion, based upon light microscopy, that successive divisions in S. aureus occur in perpendicular planes.     

Improving the Safety of Staphylococcus aureus Polyvalent Phages by Their Production on a Staphylococcus xylosus Strain

Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.     

The cell surface proteome of Staphylococcus aureus

The Gram-positive bacterium Staphylococcus aureus is a wide spread opportunistic pathogen that can cause a range of life-threatening diseases. To obtain a better understanding of the global mechanisms for pathogenesis and to identify novel targets for therapeutic interventions, the S. aureus proteome has been recently 'dissected' in several studies. Proteins that are exposed on the cell surface - collectively referred to as the 'surfacome' - have received particular attention, because they can directly interact with extracellular molecules, including drugs and antibodies. Accordingly, these proteins represent interesting candidate targets for active or passive immunization against S. aureus. Here, we review the proteomics strategies used, and we compare the results that were so far obtained. Since the surfacome is part of the cell wall proteome, we first present an overview of general properties of the S. aureus cell envelope, cell wall-associated proteins and mechanisms for protein attachment to the cell wall. Then we zoom in on the surfacome, and discuss the pro's and con's of the specific strategies that have been applied for surfacome profiling. The insights thus obtained may serve as leads for future studies on the S. aureus surfacome and possible applications.     

Interferon induced in mouse spleen cells by Staphylococcus aureus

Interferon was produced in suspensions of mouse spleen cells treated with Staphylococcus aureus preparations (killed bacteria, culture supernatants, or purified enterotoxin) under a variety of cell culture conditions. The lysate of S. aureus was found to induce high levels of interferon (10^3.1 to 10^4.3 RU/ml) within 72 hr. The crude interferon was concentrated and partially purified by either ammonium sulfate precipitation or adsorption to silicic acid and elution by ethylene glycol-containing buffer. Sequential precipitation with 50 to 80% saturated ammonium sulfate resulted in a three- to seven-fold purification with 60% recovery of activity. Adsorption to silicic acid resulted in a 25- to 80-fold purification with 77% recovery. This material was further analyzed by gel filtration. The antiviral activity induced by S. aureus-treated spleen cells was characterized as due to interferon. Furthermore, the inhibitor was acidlabile and not neutralizable by antiserum against NDV-induced L-cell interferon, thus exhibiting properties of immune (γ) interferon. The partially purified interferon was used to prepare an antiserum in rabbits. This antiserum was able to neutralize mouse interferon induced by several T-cell mitogens, by antigens, and by mixed lymphocyte cultures, while remaining inactive against interferons induced in vitro by viruses or in vivo by Brucella abortus.     

Adherence of Staphylococcus aureus to cell monolayers

Adherence of four strains of Staphylococcus aureus to eukaryotic cell monolayers was assayed with [3H]-thymidine labelled bacterial cells and the results were analysed by non-parametric statistical tests. Adherence to primary (human mesothelial) and semi-continuous (human embryonic lung) cell monolayers was significantly better than to continuous cell lines (HEp2, HeLa and Vero). HEp2 cell monolayers provided the most reliable assay substrate of the continuous cell lines tested. Variation occurred between bacterial culture batches but the assay measured significant differences between adhesion levels of the strains and distinguished between high level (RN92, 8325-4) and low level (Wood46, ISP458) adhering strains. Adherence to different batches of cell monolayers also varied but relative adherence values for strains were similar and the ranking of strains according to adhesion values was unchanged. Potential adhesion mediators have been monitored for their effect on adhesion of a highly adherent strain (RN92) to HEp2 monolayers. Fibronectin, protein A and anti-protein A did not significantly affect adhesion. Lipoteichoic acid caused a significant inhibition of adhesion. With critical statistical analysis to accommodate inherent variations, this assay provides a useful model to study factors involved in adherence of Staph. aureus to eukaryotic cells.     

Non-protein components of the cell surface of Staphylococcus aureus

1. pH–mobility curves of various laboratory strains of Staphylococcus aureus are non-sigmoid in shape, and all pass through a maximum value in the range pH4–5. 2. The maxima in the curves are not due to incomplete washing of the cells, adsorption of buffer components or irreversible surface damage. 3. Mild oxidation of the cell-surface teichoic acid with sodium metaperiodate gives cells that have typical sigmoid pH–mobility curves, characteristic of either a simple carboxyl surface or a mixed carboxyl–amino surface. 4. The results are discussed in terms of a pH-dependent change in the configuration of the teichoic acid molecules at the surface.     

Adherence of Staphylococcus aureus to cell monolayers

W yatt , J.E., P oston , S.M. & N oble , W.C. 1990. Adherence of Staphylococcus aureus to cell monolayers. Journal of Applied Bacteriology 69 , 834–844.
Adherence of four strains of Staphylococcus aureus to eukaryotic cell monolayers was assayed with [³H]-thymidine labelled bacterial cells and the results were analysed by non-parametric statistical tests. Adherence to primary (human mesothelial) and semi-continuous (human embryonic lung) cell monolayers was significantly better than to continuous cell lines (HEp2, HeLa and Vero). HEp2 cell monolayers provided the most reliable assay substrate of the continuous cell lines tested. Variation occurred between bacterial culture batches but the assay measured significant differences between adhesion levels of the strains and distinguished between high level (RN92, 8325–4) and low level (Wood46, ISP458) adhering strains. Adherence to different batches of cell monolayers also varied but relative adherence values for strains were similar and the ranking of strains according to adhesion values -was unchanged.
Potential adhesion mediators have been monitored for their effect on adhesion of a highly adherent strain (RN92) to HEp2 monolayers. Fibronectin, protein A and anti-protein A did not significantly affect adhesion. Lipoteichoic acid caused a significant inhibition of adhesion. With critical statistical analysis to accommodate inherent variations, this assay provides a useful model to study factors involved in adherence of Staph. aureus to eukaryotic cells.     

Thermogenic responses to body cooling during fever induced by Staphylococcus aureus cell walls in rabbits

Hypothalamic temperature (T _hypo) and metabolic heat production (M) were measured in seven conscious rabbits injected intravenously with either saline or with Staphylococcus aureus, (8 · 10⁷ cell walls · kg⁻¹) while being subjected to a 3-h period of ramp-like total body cooling using a chronically implanted intravascular heat exchanger. In pyrogen-injected animals cooling started (1) at the time of injection or (2) 70 min after injection. In (1) the fall in T _hypo induced by heat extraction was similar (1.0 °C) in afebrile and febrile animals. In (2) there was a transient increase in T _hypo of about 0.5 °C at a time corresponding to the start of fever resulting in a significantly smaller fall in T _hypo at the end of the 3-h cooling period (0.5 °C vs 0.9 °C, P < 0.05, n = 5). At this time in both (1) and (2) M was lower than theoretically expected from the increase in shivering threshold during fever. However, most of this effect can be explained when available data showing a decrease in thermosensitivity during S. aureus-induced fever are taken into account. After cessation of cooling in both groups of febrile animals T _hypo rose to about 1 °C higher than the precooling level, which is comparable to the fever level in a separate series of experiments with S. aureus injection without cooling (1.2 °C). Accepted: 23 September 1997     

Immunological Recognition of Fibronectin-Binding Proteins of Staphylococcus aureus and Staphylococcus capitis,Strain LK499

Antibodies to fibronectin-binding proteins (FnBPs) of Staphylococcus aureus, including binding domain of FnBPA, the D region, or the A-C regions of FnBPB were produced in rabbits and mice. These antibodies were used to characterize cell-associated FnBPs of S. aureus strain Cowan I, S. aureus strain U320 and a coagulase-negative Staphylococcus capitis strain LK499 as well as extracellular FnBPs in culture supernatants of the strain U320. FnBPs of S. aureus were predominantly FnBPA, while FnBPB was hardly detected on the cells or in culture supernatant of these S. aureus strains. Moreover, S. capitis strain LK499 possessed different FnBP(s) compared to S. aureus because the antibodies to S. aureus FnBPs did not recognize FnBP(s) on S. capitis.