Activity of the major staphylococcal autolysin Atl

The major autolysin of Staphylococcus aureus (AtlA) and of Staphylococcus epidermidis (AtlE) are well-studied enzymes. Here we created an atlA deletion mutant in S. aureus that formed large cell clusters and was biofilm-negative. In electron micrographs, the mutant cells were distinguished by rough outer cell surface. The mutant could be complemented using the atlE gene from S. epidermidis. To study the role of the repetitive sequences of atlE, we expressed in Escherichia coli the amidase domain encoded by the gene, carrying no repeat regions (amiE) or two repeat regions (amiE-R1,2), or the three repeat regions alone (R1,2,3) as N-terminal His-tag fusion proteins. Only slight differences in the cell wall lytic activity between AmiE and AmiE-R1,2 were observed. The repetitive sequences exhibit a good binding affinity to isolated peptidoglycan and might contribute to the targeting of the amidase to the substrate. AmiE and AmiE-R1,2 have a broad substrate specificity as shown by similar activities with peptidoglycan lacking wall teichoic acid, O-acetylation, or both. As the amidase activity of AtlA and AtlE has not been proved biochemically, we used purified AmiE-R1,2 to determine the exact peptidoglycan cleavage site. We provide the first evidence that the amidase indeed cleaves the amide bond between N-acetyl muramic acid and L-alanine.     

Replication of staphylococcal plasmid pT48

Insertion of a synthetic DNA linker into the repL gene of staphylococcal plasmid pT48 inactivates the replication system. This defect can be complemented in trans by the presence of a pT48 repL gene, but not by the rep genes of the related Staphylococcus areus plasmids pSN2 and pOX1000. Comparison of the sequences of the three replication proteins indicates that specificity may be determined by a putative helix-turn-helix region.     

A rapid screening procedure for staphylococcal plasmids

A method has been developed for rapidly screening representatives of all currently recognized species of the genus Staphylococcus for the presence of plasmid DNA. The isolated plasmid DNA is substantially free from contaminating chromosomal and relaxed plasmid DNA. The method will detect plasmids in strains grown on various types of solid or liquid culture media and is convenient enough for routine epidemiological studies.     

Vector engineering to improve a staphylococcal surface display system

A previously developed expression system for surface display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signals from a Staphylococcus hyicus lipase and the cell wall anchoring part of Staphylococcus aureus protein A (SpA) to achieve surface display of expressed recombinant proteins. The system has been successfully used in various applications but the vector has not been considered genetically stable enough to allow protein library display applications, which would be of obvious interest. A new set of vectors, differing in size and devoid of a phage f1 origin of replication, were constructed and evaluated in terms of bacterial growth characteristics and vector stability. Furthermore, surface expression of a model surface protein was monitored by an enzymatic whole-cell assay and flow cytometry. The engineered expression vectors demonstrated dramatically improved stability and growth properties and two of the novel vectors demonstrated retained high surface density of the displayed model protein. The flow cytometry was found to be a powerful tool for observing the surface density of displayed heterologous proteins, and would thus be a rational strategy for monitoring the optimisation of any surface display system. The implications of these improved display vectors for future protein library applications are discussed.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

Monoclonal antibodies against staphylococcal enterotoxins

MAb anti-Staphylococcal enterotoxins (MAb-SE) were produced in mice with a mixture of reference SE types A, B, C₁ and D at a concentration of 1 g each per mouse; the last booster injection was by intrasplenic route. Nine clones were chosen, two produced anti-SEB and anti-SED, one anti-SEA and anti-SEB, two anti-SED, two anti-SEB and one produced anti-SEC_1. The MAb-SE were partially purified as judged by PAGE–SDS. The partially purified antibodies could demonstrate the presence of SE in milk samples containing 0.5 g of toxin m l^–1.     

Monitoring of staphylococcal starters in two French processing plants manufacturing dry fermented sausages

AIMS: The growth and survival of Staphylococcus xylosus and Staphylococcus carnosus were monitored during sausage manufacture in two processing plants. METHODS AND RESULTS: The gram-positive, catalase-positive cocci isolated from the processing plants F10 and F11 were identified by Staphylococcus-specific PCR and species-specific oligonucleotide array. In the inoculated products with starter cultures, 90% of staphylococcal strains isolated in F10 were identified as S. xylosus and 10% as S. carnosus. In F11, 77% were identified as S. xylosus and 20% as S. carnosus. Staphylococcus xylosus dominated the staphylococcal microbiota while S. carnosus survived during the process. The pulse-field gel electrophoresis analysis revealed that all S. xylosus and S. carnosus strains isolated corresponded to the starter strains inoculated. The two starter strains of S. xylosus co-dominated in the isolates from sausages of F11, whereas the strain with pattern A1 was dominant in the isolates from sausages of F10. In the environments, no S. carnosus and S. xylosus were found, whereas Staphylococcus equorum and Staphylococcus saprophyticus were the main species isolated. CONCLUSIONS: This work highlighted the domination of S. xylosus starter strains, which showed a strong capacity to grow during sausage process, while S. carnosus survived during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Successful implantation of starter cultures is obviously a prerequisite for their contribution to sensorial qualities. Thus, the monitoring of the growth and the survival of S. xylosus and S. carnosus are required to guarantee a well-adapted starter culture. This study revealed that the two Staphylococcus species are suitable for manufacturing sausages in processing plants with very different capacities of production.     

Fluorescence studies of thermal stability of staphylococcal exfoliative toxins A and B

Abstract The quantum yields of intrinsic fluorescence of staphylococcal exfoliative toxins ETA and ETB were determined after excitation at 295 nm and 275 nm respectively. The variations in the intrinsic fluorescence and degree of fluorescence polarization showed conformational modifications of ETA around 57–59°C and of ETB around 52–54°C. Above these transition temperatures, ETB precipitated out and its biological activity was lost, whereas ETA showed almost no precipitation and no change in its biological activity, despite the structural changes in the molecule.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Cloning and expression of various staphylococcal genes encoding urease in Staphylococcus carnosus

The urease genes from Staphylococcus xylosus C2a, Staphylococcus aureus U500, and S. aureus Newman were cloned in Staphylococcus carnosus using the plasmid vectors pCA43 and pCA44. The resulting respective recombinant plasmids pUra 402, pUraUH66, and pUra17 contained chromosomal DNA fragments with sizes of 5.6, 5.8, and 6.8 kb, respectively. Investigations on urease expression of the donor and recombinant strains in media with various nitrogen sources revealed that S. xylosus C2a produced urease constitutively at the highest specific activity. All of the recombinant strains had significantly lower urease activities than their DNA-donor strains. The nickel-dependence of urease was demonstrated in S. aureus U500 by a plate diffusion assay.     

MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance

Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host's immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.     

Induction of tumour necrosis factor by staphylococcal toxic shock toxin 1

Abstract Staphylococcal toxic shock syndrome toxin 1 (TSST1) induced the release of tumour necrosis factor (TNF) from human and rabbit monocytes in vitro. Nanogram amounts of TSST1 were sufficient to induce TNF release. There was considerable variation in response between cells from different rabbits and different donors. Rabbit monocytes were slightly more sensitive to TSST1 than were human monocytes. Release of TNF in vivo could explain many of the symptoms of toxic shock syndrome.     

Bacteriocins as alternative agents for control of multiresistant staphylococcal strains

AIMS: To investigate the activity of seven staphylococcins, bacteriocins produced by staphylococci, against multiresistant Staphylococcus aureus and coagulase-negative staphylococci (CNS) involved in human infections. METHODS AND RESULTS: Four bacteriocins produced by Staph. epidermidis (Pep5, epidermin, epilancin K7 and epicidin 280) and three produced by Staph. aureus (aureocins A70, A53 and 215FN) were tested. Sixteen Staph. aureus strains, including a representative strain of the endemic Brazilian methicillin-resistant clone (MRSA), and 57 CNS strains were used as indicators. Among the staphylococcins used, Pep5 was able to inhibit 77.2% of the CNS strains and 87.5% of the Staph. aureus strains tested, including the Brazilian MRSA endemic clone, responsible for a large number of hospital-acquired infections in Brazil. On the other hand, aureocin A53 and epidermin presented a high antagonistic activity only against the Staph. aureus strains, being able to inhibit, respectively, 87.5% and 81.3% of them, including also the Brazilian MRSA endemic clone. The remaining bacteriocins inhibited only a low percentage of the nosocomial staphylococcal strains tested. CONCLUSIONS: Aureocin A53 and epidermin have potential applications against MRSA, whereas Pep5 seems to be an attractive agent against both MRSA and CNS, including mupirocin-resistant strains and the Brazilian endemic clone of MRSA, which is also found disseminated in other countries. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocins may represent alternative agents to control important nosocomial pathogens.     

Nucleotide sequence of the staphylococcal enterotoxin C3 gene sequence comparison of all three type C staphylococcal enterotoxins

Summary The structural gene entC3, which encodes staphylococcal enterotoxin C3 was cloned from the genome of Staphylococcus aureus FRI-913 and sequenced. The primary amino acid sequence of the toxin was deduced from the nucleotide sequence data. entC3 contains 801 by and encodes a precursor protein of 266 amino acids. Glutamic acid was found to be the N-terminus of mature enterotoxin C3. Thus, the first 27 residues of the toxin precursor comprise the signal peptide, and the mature toxin contains 239 amino acids with a molecular weight of 27 563 daltons. Enterotoxin C3 differs from enterotoxin C2 by four amino acids and from enterotoxin C1 by nine residues. The 167 C-terminal residues of the three toxins are identical, except for one conservative amino acid substitution in enterotoxin C3. The degree of immunological relatedness among the three Type C enterotoxins is proportional to their molecular relatedness. This study also provides evidence that the N-termini of Type C enterotoxins determine subtype-specific antigenic epitopes, while more conserved C-terminal regions determine biological properties and cross-reactive antigenic epitopes shared with other pyrogenic toxins.