EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus

Lysostaphin is an extracellular glycylglycine endopep-tidase produced by Staphylococcus simulans biovar staphylolyticus ATCC1362 that lyses staphylococcal cells by hydrolysing the polyglycine interpeptide bridges of the peptidoglycan. Renewed analysis of the sequence of the lysostaphin gene (Iss), and the sequencing of the amino-terminus of purified prolysostaphin and of mature lysostaphin revealed that lysostaphin is organized as a preproprotein of 493 amino acids (aa), with a signal peptide consisting of 36 aa, a propeptide of 211 aa from which 195 aa are organized in 15 tandem repeats of 13 aa length, and a mature protein of 246 aa. Prolysostaphin is processed in the culture supernatant of S. simulans biovar staphylolyticus by an extracellular cysteine protease. Although prolysostaphin was staphylolytically active, the mature lysostaphin was about 4.5-fold more active. The controlled expression in Staphylococcus carnosus of Iss and Iss with deletions in the prepropeptide region indicated that the tandem repeats of the propeptide are not necessary for protein export or activation of Lss, but keep Lss in a less active state. Intracellular expressed pro- and mature lysostaphin exert staphy-lolytic activity in cell-free extracts, but do not affect growth of the corresponding clones. We characterized a lysostaphin immunity factor gene (lif) which is located in the opposite direction to Iss. The expression of lif in S. carnosus led to an increase in the serine/glycine ratio of the interpeptide bridges of peptidoglycan from 2 to 35%, suggesting that lysostaphin immunity depends on serine incorporation into the interpeptide bridge. If, in addition to lif, Iss is co-expressed the serine/glycine ratio is further increased to 58%, suggesting that Lss selects for optimal serine incorporation. Lif shows similarity to FemA and FemB     

Surface disinfection properties of the combination of an antimicrobial peptide,ranalexin, with an endopeptidase,lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA)

Aims: To characterize the antibacterial synergy of the antimicrobial peptide, ranalexin, used in combination with the anti‐staphylococcal endopeptidase, lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA), and to assess the combination’s potential as a topical disinfectant or decolonizing agent for MRSA. MRSA causes potentially lethal infections, and pre‐operative patients colonized with MRSA are often treated with chlorhexidine digluconate and mupirocin cream to eradicate carriage. However, chlorhexidine is unsuitable for some patients, and mupirocin resistance is increasingly encountered, indicating new agents are required. Methods and Results: Using an ex vivo assay, ranalexin and lysostaphin tested in combination reduced viable MRSA on human skin to a greater extent than either compound individually. The combination killed bacteria within 5 min and remained effective and synergistic even in high salt and low pH conditions. Conclusions: The combination is active against MRSA on human skin and under conditions that may be encountered in sweat. Significance and Impact of the Study: Although the exact mechanism of activity remains unresolved, considering its specific spectrum of activity, fast killing kinetics and low likelihood of resistance arising, the combination of ranalexin with lysostaphin warrants consideration as a new agent to eradicate nasal and skin carriage of Staph. aureus, including MRSA.     

Staphylococcus aureus Response to Lysostaphin in Some Fermented Foods

The addition of lysostaphin to starting materials for cheese and fermented sausage that were artificially contaminated with Staphylococcus aureus resulted in an initial decrease in the staphylococcal flora. In a simulated cheese process, lysostaphin remained with the curd after separation of the whey. In both cheese and fermented sausage samples that were produced experimentally in the laboratory, a significant S. aureus population ultimately developed, even in the presence of lysostaphin. Staphylococcal isolates from these treated products were not more resistant to the lytic enzyme than was the parent strain.     

Cost-effective optimization of gellan gum production by Sphingomonas paucimobilis using corn steep liquor

Abstract

Gellan gum, produced by Sphingomonas paucimobilis, is increasingly used in food and pharmaceutical industries as stabilizing, emulsifying, texturing and gelling agents. However, its high production costs may limit its full commercial potential. Therefore, in this study, we investigated ways to reduce gellan gum production costs and improve yields. We first revealed corn steep liquor (CSL) as a cost-effective nutrient source that can improve gellan gum yields. We then systematically optimized culture conditions even further, and revealed that the addition of Triton X-100 surfactant and selected inorganic nitrogen sources improved gellan gum production. Under our optimized conditions (glucose 33.75?g/L, CSL 10?g/L, urea 2.5?g/L, MgSO₄ 1.08?g/L, KH₂PO₄ 3.24?g/L, K₂SO₄ 1?g/L and Triton X-100 0.75?g/L), we yielded a maximum concentration of 14.41?g/L, which was about 1.5-fold higher than non-optimized CSL-based medium. Our findings highlight the use of CSL as a cost effective and promising nutrient source for industrial production of gellan gum.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Therapeutic applications of lysostaphin against Staphylococcus aureus

Staphylococcus aureus, an opportunistic pathogen, causes diverse community and nosocomial-acquired human infections, including folliculitis, impetigo, sepsis, septic arthritis, endocarditis, osteomyelitis, implant-associated biofilm infections and contagious mastitis in cattle. In recent days, both methicillin-sensitive and methicillin-resistant S. aureus infections have increased. Highly effective anti-staphylococcal agents are urgently required. Lysostaphin is a 27 kDa zinc metallo antimicrobial lytic enzyme that is produced by Staphylococcus simulans biovar staphylolyticus and was first discovered in the 1960s. Lysostaphin is highly active against S. aureus strains irrespective of their drug-resistant patterns with a minimum inhibitory concentration of ranges between 0·001 and 0·064 μg ml⁻¹. Lysostaphin has activity against both dividing and non-dividing S. aureus cells; and can seep through the extracellular matrix to kill the biofilm embedded S. aureus. In spite of having excellent anti-staphylococcal activity, its clinical application is hindered because of its immunogenicity and reduced bio-availability. Extensive research with lysostaphin lead to the development of several engineered lysostaphin derivatives with reduced immunogenicity and increased serum half-life. Therapeutic efficacy of both native and engineered lysostaphin derivatives was studied by several research groups. This review provides an overview of the therapeutic applications of native and engineered lysostaphin derivatives developed to eradicate S. aureus infections.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the spheroplast formation from these bacteria.     

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A+T/G+C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel high-level expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants.     

Biologic Activity of Cell-Associated Staphylococcal Enterotoxin A

Cell associated staphylococcal enterotoxin A, released by lysostaphin treatment of Staphylococcus aureus cells, was found to be biologically active in cynomolgus monkeys. The activity was comparable to that of extracellular enterotoxin A; six of six monkeys vomited within 5 h in response to extracellular enterotoxin and four of six also vomited in response to the same serologic level (4.8 μg per monkey) of cell-associated enterotoxin. Feeding S. aureus cells containing cell-associated enterotoxin A to cynomolgus monkeys resulted in emesis in three of five monkeys within 3 h. This suggests that consumption of S. aureus cells could lead to staphylococcal intoxication.     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Influence of Lif, the Lysostaphin Immunity Factor, on Acceptors of Surface Proteins and Cell Wall Sorting Efficiency in Staphylococcus carnosus

Proteins harboring a C-terminal cell wall sorting signal are covalently linked to pentaglycine acceptors within the staphylococcal peptidoglycan. This pentaglycine was modified when the lysostaphin immunity factor (Lif) of Staphylococcus simulans was expressed in Staphylococcus carnosus, likely by the exchange of two glycine residues for serine residues. A reporter protein was efficiently linked to the modified acceptor, indicating that the sorting reaction is not strictly dependent on the wild-type structures of the acceptors.     

Characterization of the staphylococcal bacteriophage lysin CHAP(K)

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAP_K. To evaluate its antibacterial spectrum_, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAP_K was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP_K includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP_K and lysostaphin was compared over a concentration range of 2·5–10 μg ml^?1 using live Staph. aureus for 5 min at 37°C, CHAP_K gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage‐derived enzyme CHAP_K demonstrating that it has excellent biochemical properties as an anti‐staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP_K as an alternative therapeutic against pathogenic staphylococci including MRSA.     

Supplementation of watermelon peels as an enhancer of lipase and esterase production by Yarrowia lipolytica in solid-state fermentation and their potential use as biocatalysts in poly(ethylene terephthalate) (PET) depolymerization reactions

Abstract

Yarrowia lipolytica is a yeast that presents high biotechnological potential due to its ability to produce many metabolites, among them lipases and esterases, which are important industrial biocatalysts. Since Brazil is an agroindustrial country, it generates an enormous diversity of residues or byproducts that can be used as a platform for biomolecules production. This work aims to evaluate lipase and esterase production by Y. lipolytica via solid-state fermentation using soybean bran and soybean bran supplemented with watermelon peels in different contents, and subsequent use of the enzyme extracts for poly(ethylene terephthalate) (PET) hydrolysis. Supplementation of watermelon peels in the lowest content led to an improvement of lipase activity in almost 31%, reaching 75.22?U g^?1. Esterase productivity was 1.5-fold higher when 20?wt% of watermelon peels were added to the media culture. Timecourse evaluation of enzymes production showed a maximum lipase activity in 14?h and similar esterase activity in 14 and 20?h of fermentation. Proteases production were also intensified in supplemented samples. Enzymes produced with 5?wt% watermelon peels supplementation led to higher terephthalic acid concentration (up to 42.02?µmol L^?1) during PET depolymerization. Results suggest a great potential of enzyme production in low cost fermentative media to act as biocatalysts in PET hydrolysis reactions.     

Tolerance of Landoltia punctata to arsenate: an evaluation of the potential use in phytoremediation programs

Abstract

Plants used in phytoremediation should accumulate and tolerate a specific pollutant. Here, we aimed at evaluating a possible arsenic (As) accumulation and mechanisms of tolerance against As-induced damage in Landoltia punctata to explore this species for phytoremediation. Plants were subjected to increasing As levels. As absorption was higher with increasing As levels. The activity of superoxide dismutase and glutathione reductase as well as anthocyanin levels increased with As levels. Catalase and peroxidase activities increased in plants subjected to As levels up to 1.0?mg L^?1 and decreased at higher levels. Due to the antioxidant system, higher levels of reactive oxygen species were restrained in plants under low levels of As. However, the levels of superoxide anion, hydrogen peroxide, and lipid peroxidation increased in response to the impaired antioxidant system induced by the highest As levels. Biomass decreased in plants exposed to As and scanning electron microscopy revealed root structural damage in the root cap of plants under 3.0?mg L^?1?As. This work highlights that L. punctata can be considered a hyperaccumulator species and has potential for As phytoremediation when levels are lower than 1.0?mg L^?1—a concentration 100-fold higher than that recommended for drinking water.

Novelty Statement: Landoltia punctata can be considered a hyperaccumulator species and has the potential for arsenic phytoremediation when levels are lower than 1.0?mg L^?1.     

Cloning, Expression, and Purification of the Staphylococcus simulans Lysostaphin Using the Intein-Chitin-Binding Domain (CBD) System

The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source

Abstract

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9?±?43.8?mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.     

Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives

The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.