Characterization of a Staphylococcus aureus Bacteriocin

The bacteriocin produced by a strain of Staphylococcus aureus has been isolated and designated staphylococcin (414), and a study was made of its chemical, physical, and biological properties. The staphylococcin is released in appreciable quantities after breakage of the cells and can be purified through differential centrifugation and column chromatography. In the native state, it appears to be a lipoprotein-carbohydrate complex with a molecular weight in excess of 200,000. The complex can be dissociated by sodium dodecyl sulfate into smaller subunits which retain activity. The gross chemical and physical properties of the bacteriocin closely resemble those ascribed to certain preparations of cell membranes. Staphylococcin (414) is not a lytic enzyme like lysostaphin and does not have the same spectrum of activity. Like other bacteriocins from gram-positive microorganisms, it does not inhibit any gram-negative bacteria, but does inhibit several other genera.     

Characterization of a temperature-sensitive DNA replication mutant of Staphylococcus aureus

A temperature-sensitive DNA replication mutant of Staphylococcus aureus NCTC 8325 has been isolated and characterized. After transfer to the non-permissive-temperature (42 degrees C), DNA synthesis continued for 30 min and the mean DNA content increased by 56%. The amount of residual DNA synthesis was not reduced when the non-permissive temperature was raised, nor when chloramphenicol was added at the time of the temperature shift. During incubation at 42 degrees C, mutant bacteria accumulated the capacity to synthesize DNA after return to the permissive temperature (30 degrees C) in the presence of chloramphenicol. This capacity was lost when chloramphenicol was present at 42 degrees C. The properties of the mutant are consistent with a defect in the initiation of DNA replication at 42 degrees C.     

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.     

Characterization of Staphylococcus aureus SarA binding sites

The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.     

Characterization of RAP,a quorum sensing activator of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria and cause diverse serious diseases in humans and animals through the production of toxins. The production of toxins is regulated by quorum sensing mechanisms, where proteins such as RNAIII activating protein (RAP) are secreted by the bacteria and induce virulence. Antibodies to RAP have been shown to protect mice from infection, but the molecular structure of RAP was not known and hindered vaccine development. To characterize RAP, recombinant protein was made and tested for its ability to induce genes important for pathogenesis (agr). In addition, monoclonal antibodies were produced to identify its cellular localization. Results shown here indicate that RAP is a 277-aa protein that is an ortholog of the ribosomal protein L2. Like the native molecule, recombinant RAP activates the production of RNAIII (encoded by agr). Using RAP specific monoclonal antibodies we demonstrate that RAP is continuously secreted and while RAP is expressed also in other bacteria (like Staphylococcus epidermidis, Staphylococcus xylosus and Escherichia coli), it is secreted to the culture medium only by S. aureus. Our results show that the ribosomal protein L2 has an extraribosomal function and that when secreted RAP acts as an autoinducer of virulence to regulate S. aureus pathogenesis.     

Characterization of the Starvation-Survival Response of Staphylococcus aureus

The starvation-survival response of Staphylococcus aureus as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [³⁵S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.     

Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus

Staphylococcal nuclease (here termed as Nuc1) is considered an important virulence factor and a unique marker widely used in the detection of Staphylococcus aureus. A second functional thermostable nuclease (here termed as Nuc2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nuc1. Recombinant Nuc2 showed nuclease activity in the zymogram test and was able to degrade various types of nucleic acids. The optimal reaction temperature and pH for Nuc2 were 50 °C and pH 10, respectively. The enzymatic activity of Nuc2 was stimulated in the presence of Ca²⁺ (0.05 mM), Mg²⁺ (0.5 mM), dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn²⁺ and Mn²⁺, and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nuc1, which was consistent with differences in the sequence pattern and structure predicted. Furthermore, a nuc1 and nuc2 double deletion mutant of S. aureus and respective complementation experiments suggest a major role for nuc1 in terms of thermonuclease activity in S. aureus.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Characterization of Chloramphenicol Acetyltransferase from Chloramphenicol-resistant Staphylococcus aureus

Chloramphenicol-resistant strains of Staphylococcus aureus contain an inducible enzyme which inactivates chloramphenicol by acetylation in the presence of acetyl coenzyme A. The products of acetylation are chromatographically indistinguishable from those obtained with chloramphenicol-resistant Escherichia coli harboring an R factor. The kinetics of induction of chloramphenicol acetyltransferase are complicated by the inducer's effect on protein biosynthesis and its fate as chloramphenicol 3-acetate, which is not an inducer of the enzyme. The E. coli and S. aureus enzymes have been compared, with the conclusion that they are identical with respect to molecular weight (approximately 78,000) and pH optimum (7.8), but differ with respect to heat stability, substrate affinity, electrophoretic mobility, and immunological reactivity. Antiserum prepared against enzyme from E. coli contains precipitating antibody, which inactivates the E. coli enzyme, but neither precipitates nor neutralizes the activity of S. aureus enzyme.     

Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis

Staphylococcus aureus is an important pathogen that continues to be a significant global health threat because of the prevalence of methicillin-resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defence against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal host. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis.     

Characterization of hemin antibacterial action on Staphylococcus aureus

Abstract The mechanism of inactivation of Staphylococcas aureus cells by hemin is described. Protection experiments by sulfhydryl reagents such as cysteine, mercaptoethanol, glutathione or thioglycolate in their reduced form prevent S. aureus bacteria from inactivation by hemin (1.5 × 10⁻⁵ M). The treatment of bacteria by hemin in the presence of one of those reagents (1 × 10⁻² M) showed that the growth rate and viability of the culture remained unchaged. On the other hand sulfhydryl reagents did not prevent the binding of hemin to the bacteria. When cysteine or glutathione were introduced to a culture after exposure to hemin it could neither reverse the damage done to the cells nor shorten the time of the culture's recovery. Another type of protection was obtained by addition of serum albumin which prevented hemin molecules from binding to the bacterial envelopes. Furthermore, when albumin was introduced after the bacteria were treated by hemin it prevented further damage to the survivors and thus shortened the time required for recovery. None of the singlet oxygen quenchers or hydroxyl radical scavengers could protect the bacteria from hemin inactivation. The mechanism by which hemin affects S. aureus is assumed to be by oxidizing a major system within the cell.     

Emergence of Community-Associated Methicillin-Resistant Staphylococcus aureus Associated with Pediatric Infection in Cambodia

Background

The incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection is rising in the developed world but appears to be rare in developing countries. One explanation for this difference is that resource poor countries lack the diagnostic microbiology facilities necessary to detect the presence of CA-MRSA carriage and infection.

Methodology and Principal Findings

We developed diagnostic microbiology capabilities at the Angkor Hospital for Children, Siem Reap, western Cambodia in January 2006 and in the same month identified a child with severe community-acquired impetigo caused by CA-MRSA. A study was undertaken to identify and describe additional cases presenting between January 2006 and December 2007. Bacterial isolates underwent molecular characterization using multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the presence of the genes encoding Panton-Valentine Leukocidin (PVL). Seventeen children were identified with CA-MRSA infection, of which 11 had skin and soft tissue infection and 6 had invasive disease. The majority of cases were unrelated in time or place. Molecular characterization identified two independent MRSA clones; fifteen isolates were sequence type (ST) 834, SCCmec type IV, PVL gene-negative, and two isolates were ST 121, SCCmec type V, PVL gene-positive.

Conclusions

This represents the first ever report of MRSA in Cambodia, spread of which would pose a significant threat to public health. The finding that cases were mostly unrelated in time or place suggests that these were sporadic infections in persons who were CA-MRSA carriers or contacts of carriers, rather than arising in the context of an outbreak.     

Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus

Plasmids specifying bacteriocin production and immunity to its action were found in three clinical isolates of Staphylococcus aureus obtained in different hospitals located in Rio de Janeiro. These plasmids (pRJ28, pRJ29 and pRJ30) of 8.0 kb were found to generate identical restriction fragment patterns upon digestion with several enzymes, although the range of strains susceptible to the respective bacteriocin varied among the producer strains, when different Gram-positive bacteria were used as indicators. pRJ29 was then chosen for further characterization in order to compare it with pRJ6 and pRJ9, two small bacteriocin-encoding plasmids previously described in strains isolated from food. pRJ29 was found to code for a bacteriocin with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis, and estimated molecular weight) similar to those of pRJ6-encoded bacteriocin, conferring cross-immunity to it. However, its restriction map differed from those of pRJ6 and pRJ9. These studies together with hybridization, incompatibility, and mobilization analyses using a derivative of pRJ29 tagged with Tn917-lac suggest that pRJ29 is a mosaic composed of genetic determinants found on pRJ6 and pRJ9, and that IS 257 was not involved in the recombination events which gave rise to pRJ29.     

Characterization of RAT,an autolysis regulator in Staphylococcus aureus

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.