Staphylococcus aureus nuclease is an SaeRS-dependent virulence factor.

Several prominent bacterial pathogens secrete nuclease (Nuc) enzymes that have an important role in combating the host immune response. Early studies of Staphylococcus aureus Nuc attributed its regulation to the agr quorum-sensing system. However, recent microarray data have indicated that nuc is under the control of the SaeRS two-component system, which is a major regulator of S. aureus virulence determinants. Here we report that the nuc gene is directly controlled by the SaeRS two-component system through reporter fusion, immunoblotting, Nuc activity measurements, promoter mapping, and binding studies, and additionally, we were unable identify a notable regulatory link to the agr system. The observed SaeRS-dependent regulation was conserved across a wide spectrum of representative S. aureus isolates. Moreover, with community-associated methicillin-resistant S. aureus (CA MRSA) in a mouse model of peritonitis, we observed in vivo expression of Nuc activity in an SaeRS-dependent manner and determined that Nuc is a virulence factor that is important for in vivo survival, confirming the enzyme's role as a contributor to invasive disease. Finally, natural polymorphisms were identified in the SaeRS proteins, one of which was linked to Nuc regulation in a CA MRSA USA300 endocarditis isolate. Altogether, our findings demonstrate that Nuc is an important S. aureus virulence factor and part of the SaeRS regulon.

Production and purification of staphylococcal nuclease in Lactococcus lactis using a new expression-secretion system and a pH-regulated mini-reactor.

BACKGROUND: Staphylococcal (or micrococcal) nuclease or thermonuclease (SNase or Nuc) is a naturally-secreted nucleic acid degrading enzyme that participates in Staphylococcus aureus spread in the infected host. Purified Nuc protein can be used as an exogenous reagent to clear cellular extracts and improve protein purification. Here, a recombinant form of Nuc was produced and secreted in a Gram-positive host, Lactococcus lactis, and purified from the culture medium. RESULTS: The gene segment corresponding to the S. aureus nuclease without its signal peptide was cloned in an expression-secretion vector. It was then fused to a lactococcal sequence encoding a signal peptide, and expressed under the control of a lactococcal promoter that is inducible by zinc starvation. An L. lactis subsp cremoris model strain (MG1363) transformed with the resulting plasmid was grown in either of two media (GM17v and CDM) that are free of animal compounds, allowing GMP (Good Manufacturing Practice) production. Induction conditions (concentration of the metal chelator EDTA and timing of addition) in small-scale pH-regulated fermentors were optimized using LacMF (Lactis Multi-Fermentor), a home-made parallel fermentation control system able to monitor 12 reactors simultaneously. Large amounts of recombinant Nuc (rNuc) were produced and secreted in both media, and rNuc was purified from GM17v medium in a single-step procedure. CONCLUSIONS: In L. lactis, rNuc production and secretion were optimal after induction by 0.5 mM EDTA in small scale (200 mL) GM17v exponential phase cultures (at an OD(600) of 2), leading to a maximal protein yield of 210 mg per L of culture medium. Purified rNuc was highly active, displaying a specific activity of 2000 U/mg.

Highly efficient production of the staphylococcal nuclease reporter in Lactobacillus bulgaricus governed by the promoter of the hlbA gene.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) genome sequence analysis revealed the presence of two genes that encode histone-like HU proteins (hlbA and hlbB) showing extensive similarity to other bacterial homologues. These genes were found to be extremely conserved among several L. bulgaricus strains. The hlbA gene was shown to be constitutively transcribed from a unique promoter (phlbA) during normal growth, whereas hlbB did not seem to be expressed under usual laboratory conditions. Using a reporter cassette in which the staphylococcal nuclease was fused at its N-terminus to the lactococcal signal peptide Usp45 (SP Usp45), we have demonstrated that phlbA promotes high expression of the reporter in L. bulgaricus, which correlated with an abundant secretion of the mature nuclease in the supernatant fraction. Quantification of the exported enzyme reveals a secretion level approximately threefold higher when the expression of the reporter was under the control of phlbA compared with the lactococcal usp45 promoter. Together, these results indicate that phlbA is suitable for gene expression in L. bulgaricus, that SP Usp45 is functionally recognized and processed by the L. bulgaricus secretion machinery and that the nuclease reporter gene can be used for the identification of exported products in this bacterium.

[Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests]. / Deteccion de la enterotoxina A de Staphylococcus aureus mediante la reaccion en cadena de la polimerasa (PCR) y su correlacion con las pruebas de coagulasa y termonucleasa.

Staphylococcus aureus is a pathogenic bacterium, widely distributed on nature and associated to general infection and food borne outbreaks. The relationship between this bacterium and food borne outbreaks has been done, historically, using several tests, including coagulase, thermonuclease and actually, PCR for the genes codifying for the enterotoxin responsible of clinical symptoms. The objective of this work is to detect enterotoxin A codifying gene through PCR in a group of S. aureus strains isolated from food samples, and also to correlate the presence of this gene with the production of coagulase and thermonuclease enzymes. A total of 69 staphylococcal strains were analyzed, 58 obtained from non pasteurized milk samples from the Estación Experimental Alfredo Volio Mata and 11 from the Food and Water Microbiology Laboratory collection, Universidad de Costa Rica. Coagulase, thermonuclease and enterotoxin A were analyzed in all the strains, and a statistical correlation was performed in order to verify possible associations. Results show that there is no correlation between the three variables, nevertheless, all coagulase positive strains were thermonuclease positive, and all enterotoxin positive strains were coagulase and thermonuclease positive, but not inversely. These results show that the use of presumptive or indirect tests for establishing entorotoxigenity of S. aureus strains is not truthful, more sensible and specific analysis, as PCR, shall be performed.

[Capsid-targeted viral inactivation for dengue virus infection].

To explore the feasibility of capsid-targeted viral inactivation for dengue virus infection, a newly-discovered antiviral strategy, a mammalian cell line stably expressing staphylococcal nuclease fused to the capsid protein of dengue 2 virus was established and the effects on the production of infectious virus particles were examined. The results presented evidence that the enzymatically active staphylococcal nuclease fused to capsid protein could be incorporated into the nascent virions during wild virus assembly, resulting in degradation of viral genomic RNA and decrease in infectivity. Comparing the effects of incorporated SN and SN*, an enzymatically inactive missense mutant form of SN, on the infectivity of progeny virions, nucleolytic activity of incorporated SN was responsible for the major antiviral effects. These results paved the road of developing capsid-targeted viral inactivation as a new antiviral strategy against dengue.

Influence of lipoteichoic acid D-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis.

Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in L. lactis. Random insertional mutagenesis was performed with L. lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L. lactis (approximately 40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.

[Evaluation of the effect of probiotic cultures on two different yogurt brands over a known population of Staphylococcus aureus and the production of thermonuclease]. / Evaluación del efecto de cultivos probióticos presentes en yogurt sobre Staphylococcus aureus y la producción de termonucleasa.

The effect of probiotic cultures over known populations of Staphylococcus aureus inoculated in yogurt was studied; also the production and stability of its thermonuclease during yogurt storage was evaluated. In three different occasions, two different yogurt brands, one with additional probiotic cultures (Lactobacillus casei and L. acidophilus), were inoculated with known populations of S. aureus in high and low concentration (10(9) CFU/g and 10(7) CFU/g), respectively. These samples were stored for 28 days at 5 degrees C. Every four days the count of lactic bacteria, S. aureus and pH were evaluated, according to the methodology described in the Compendium of Methods for the Microbiological Examination of Foods, Vanderzant & Splittstoesser. The presence of thermonuclease was determined using petrifilm for S. aureus from 3M company. The pH and lactic bacteria population were constant during the testing period. Yogurt with additional probiotic cultures (high and low concentration) lowered the population of S. aureus to non detectable levels in 8 days; but, S. aureus could be cultured from yogurt without probiotics even after 24 days of incubation. Same time, the presence of thermonuclease was positive in all tests; it was not affected by probiotics. The presence of thermonuclease is related to the production of S. aureus enterotoxin. This work emphasizes again the beneficial effects of probiotic cultures in yogurt over bacteria and the importance of keeping hygienic practices in order to avoid the contamination of food with S. aureus and the eventual production of its enterotoxin, since it is not affected by probiotics.

Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis.

The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.

A modified Escherichia coli protein production strain expressing staphylococcal nuclease, capable of auto-hydrolysing host nucleic acid.

The large-scale production of recombinant biotherapeutics, particularly recombinant proteins, provides significant process and regulatory challenges to the biotechnology industry in order to meet the regulatory agencies stringent requirements in a cost-effective manner. Host cell derived nucleic acid causes problems from both a process and a regulatory perspective, as high molecular weight chromosomal DNA is responsible both for the viscosity of cell lysates, and it is a source of heterologous DNA sequences whose inclusion in the final product must be prevented. We have constructed a modified Escherichia coli JM107 expression host (JMN), containing a staphylococcal nuclease expression cassette, integrated into the host chromosome at the dif locus. The nuclease is expressed as a fusion to the ompA signal peptide, and is translocated to the periplasm of the cell, protecting the cytoplasmic nucleic acid from any toxic activity. The nuclease is released during cell lysis, where it subsequently acts to hydrolyse host nucleic acid present in the lysate. Results with this strain show that sufficient levels of nuclease activity are produced to completely auto-hydrolyse the host's chromosomal DNA to a size non-visible on 1% agarose gel, generating a markedly lower lysate viscosity. This provides a suitable methodology to remove heterologous DNA sequences early in the product stream and decrease lysate viscosity, improving the efficiency of downstream processing and product yield, whilst avoiding the addition of exogenous nuclease and its prohibitive costs at large-scale.

The effect of Nepeta cataria extract on adherence and enzyme production of Staphylococcus aureus.

Nepeta cataria L., commonly known as catnip, is a perennial herb with a considerable folkloric reputation. A diethyl ether extract of this plant has been shown to have antimicrobial activity against fungi and Gram-positive bacteria. The aim of this work was to study the activity of N. cataria extract on 44 Staphylococcus aureus strains, some resistant to methicillin, and S. aureus 6538P (American Type Culture Collection) by evaluating the effect of subminimum inhibitory concentrations on coagulase, DNAse, thermonuclease and lipase production, and on in-vitro adherence. DNAse, thermonuclease and lipase were inhibited by concentrations equal to 1/2 and 1/4 MIC. A reduction of adherence was also observed.

Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system.

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small ( approximately 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus. A deletion of amino acids 114-119 causes monomeric NucA to form homodimers. The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay. We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium. The activity of the dimer NucA was approximately 100-fold less than the monomer. Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at approximately 18 or approximately 30 kDa, respectively. In addition, similar to S. aureus NucA, the S. gordonii recombinant NucA enzyme was dependent on CaCl(2) and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX. This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines.

Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin.

Brazzein is a 54-amino-acid sweet-tasting protein first isolated from the fruit of Pentadiplandra brazzeana Baillon found in West Africa. Brazzein, as isolated from the fruit, is 500 times sweeter than sucrose on a weight basis (9500 times sweeter on a per-molecule basis). A minor component of brazzein from fruit, des-pGlu1-brazzein, has 53 amino acid residues and has twice the sweetness of the parent protein. We have designed a gene for des-pGlu1- brazzein that incorporates codons that are optimal for protein production in Escherichia coli. Production of brazzein from the chemically synthesized gene resulted in recombinant protein with sweetness similar to that of brazzein isolated from the original source. The best yields were achieved by producing brazzein as a fusion with staphylococcal nuclease with a designed cyanogen bromide cleavage site. Because of its intense sweetness and stability at high pH and temperature, brazzein is an ideal system for investigating the chemical and structural requirements involved in sweet-taste properties. This efficient protein production system for brazzein will facilitate such investigations.

Folding of SNase R begins early during synthesis: the conformational feature of two short N-terminal fragments of staphylococcal nuclease R.

To further understand the folding of nascent peptide during the early course of peptide synthesis, two short N-terminal fragments of staphylococcal nuclease R (SNase R), SNR52 and SNR79, were made by deleting 97 and 70 amino acid residues from the C-terminus. The conformations of SNR52 and SNR79 were studied by FTIR and far-ultraviolet CD. The results demonstrate that even the short N-terminal fragments of SNase R still have a certain amount of residual ordered secondary structure in the physiological condition. The ordered secondary structures were mainly assigned as beta-strands and turns, which corresponds well to the structures of the N-terminal part in the native protein. The conformational changes during unfolding and refolding in different concentrations of guanidine hydrochloride (GuHCl), monitored by far-ultraviolet CD and intrinsic fluorescence, show that the interaction between amino acid residues, which governs the formation of their conformation are not random. Considered together with earlier studies (Jing et al., Biochim Biophys Acta 1995;1250:189-196; Zhou et al., J Biochem 1996:120: 881-888), the results suggest that the folding of nascent peptide chains begins early in the synthesis process and that the amount of ordered structure increases with increasing peptide chain length until the conformation of the biologically active protein is generated.

Triple-resonance NOESY-based experiments with improved spectral resolution: applications to structural characterization of unfolded, partially folded and folded proteins.

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C' (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C' resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in 1H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C' either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), delta 131 delta, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate H alpha protons or protons resonating close to water.

Effect of three commercial starters on growth of Staphylococcus aureus and enterotoxins (A-D) and thermonuclease production in broth.

The growth of four enterotoxigenic Staphylococcus aureus strains was partially inhibited by three commercial starters used in the meat sausage industry when grown in APT broth at 30 degrees C statically. Starter SP318 (a mixture of selected strains of Lactobacillus sake, Pediococcus pentosaceus and Staphylococcus xylosus) showed the most inhibitory activity. Staphylococcal enterotoxins (A, B, C1 and D) synthesis was totally inhibited by the growth of the three starters, whereas staphylococcal thermonuclease production was partially inhibited in mixed cultures.

Staphylococcal growth and enterotoxins (A-D) and thermonuclease synthesis in the presence of dehydrated garlic.

The inhibition of Staphylococcus aureus growth and enterotoxin and thermonuclease production by various concentrations of garlic (Allium sativum) was studied in BHI broth. The growth of Staph. aureus was inhibited by dehydrated garlic at levels of 1.5% (w/v) and over. Enterotoxins A, B and C1 were only detectable in broth containing < 1% of garlic while enterotoxin D was produced at a level of 2%. Garlic also inhibited thermonuclease (TNAse) production, complete inhibition being observed at levels > or = 1.5%. TNAse was not always detected when enterotoxin was present.

Engineering alternative beta-turn types in staphylococcal nuclease.

We have refined the crystal structures of three point mutants of staphylococcal nuclease designed to favor alternative beta-turn types. Single amino acid substitutions were made in a type VIa beta-turn (residues 115-118; Tyr-Lys-Pro-Asn) containing a cis Lys 116-Pro 117 peptide bond. The mutations result in two new backbone conformations, a type I beta-turn for P117T and a type I' beta-turn for P117G and P117A. The P117G and P117A structures exhibit a dramatic difference in backbone conformation in the region of the mutation compared to the nuclease A structure such that the side chain of Lys 116 is reoriented to point into the nucleotide binding pocket. The distinct conformation observed for the nuclease A, P117G, and P117T beta-turn sequences agrees with correlations between beta-turn type and sequence identified from protein crystal structures. The P117A turn conformation provides an exception to these correlations. The results demonstrate that single residue changes can significantly alter backbone conformation, illustrating the process by which diversity in the structure of the protein surface can evolve on a conserved structural core, and suggest protein engineering applications in which the positioning as well as the identify of side chains can be modified to design new enzyme functions. Nuclease variants at the type VIa beta-turn site also allow the relationship between the amino acid sequence and beta-turn conformation to be examined in the context of an identical protein fold in crystallographic detail.

High-level expression of staphylococcal nuclease A in Escherichia coli.

The staphylococcal nuclease A gene has been successfully cloned and overexpressed in E. coli under the transcriptional control of the bacteriophage lambda PRPL promoters regulated by the temperature sensitive repressors. The SDS-PAGE analysis demonstrates that the nuclease A is produced to the extent of as much as 60% of the total cellular protein. The N-terminal analysis of the nuclease A shows that the amino terminal formyl methionine residue of the enzyme is precisely processed. The recombinant nuclease A with full activity is finally obtained after appropriate solubilization--denaturation and renaturation treatment. The conformational identity of the renatured nuclease A in different conditions is also studied by using hydrophobic interaction chromatography on a phenyl-superose HR5/5 column.

Temperature limits of growth, TNase and enterotoxin production of Staphylococcus aureus strains isolated from foods.

For 77 strains of Staphylococcus aureus freshly isolated from different foods, growth, enterotoxin and TNase production were determined in intervals of 1.5 degrees C +/- 0.5 degrees C by cultivating them in a temperature-gradient incubator between 5 and 50 degrees C for up to 7 days. All the strains were coagulase, DNase and lysostaphin positive but only 58% formed one or two enterotoxins type SEA, SEB or SEE. All strains grew within 7 days in brain heart infusion and had lower and upper temperature limits for growth and TNase production of between 6.5 and 12.5 degrees C, and 39.5 and 48.5 degrees C respectively. The lower and upper temperature limits for production of enterotoxins were between 14 and 38 degrees C, and between 35 and 44 degrees C respectively. Enterotoxin forming isolates either showed narrow (3 to 4 degrees C) or wide (10 to 20 degrees C) ranges of enterotoxin production, irrespective of their temperature range of growth and TNase production. None of the 12 specific physiological attributes used for differentiation could be correlated to toxin type or the temperature requirement of the toxin production. No correlation between the origin and the physiological characters could be detected.