High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage.

CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif. We transferred LivCRISPR-1 cas9 and trans-activating crRNA into Listeria monocytogenes. Along with crRNA encoding plasmids, this programmable interference system enables efficient cleavage of bacterial DNA and incoming phage genomes. We used LivCRISPR-1 to develop an effective engineering platform for large, non-integrating Listeria phages based on allelic replacement and CRISPR-Cas-mediated counterselection. The broad host-range Listeria phage A511 was engineered to encode and express lysostaphin, a cell wall hydrolase that specifically targets Staphylococcus peptidoglycan. In bacterial co-culture, the armed phages not only killed Listeria hosts but also lysed Staphylococcus cells by enzymatic collateral damage. Simultaneous killing of unrelated bacteria by a single phage demonstrates the potential of CRISPR-Cas-assisted phage engineering, beyond single pathogen control.

Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn2+ on bacteriolytic activity.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn2+, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn2+-chelator), the adverse effect of the exogenous Zn2+ on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn2+ from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn2+-binding site found in close proximity to the Zn2+-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn2+ ions on staphylolytic activity of the purified Zn2+-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.

Staphylococcus simulans 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 Sigma-Aldrich (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.

Zinc-finger nickase-mediated insertion of the lysostaphin gene into the beta-casein locus in cloned cows.

Zinc-finger nickases (ZFNickases) are a type of programmable nuclease that can be engineered from zinc-finger nucleases to induce site-specific single-strand breaks or nicks in genomic DNA, which result in homology-directed repair. Although zinc-finger nuclease-mediated gene disruption has been demonstrated in pigs and cattle, they have not been used to target gene addition into an endogenous gene locus in any large domestic species. Here we show in bovine fetal fibroblasts that targeting ZFNickases to the endogenous ß-casein (CSN2) locus stimulates lysostaphin gene addition by homology-directed repair. We find that ZFNickase-treated cells can be successfully used in somatic cell nuclear transfer, resulting in live-born gene-targeted cows. Furthermore, the gene-targeted cows secrete lysostaphin in their milk and in vitro assays demonstrate the milk's ability to kill Staphylococcus aureus. Our success with this strategy will facilitate new transgenic technologies beneficial to both agriculture and biomedicine.

[Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin].

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.

[Purification of recombinant lysostaphin by monoclonal antibody affinity chromatography].

Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established the monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.

New effective sources of the Staphylococcus simulans lysostaphin.

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.

Expression of lysostaphin in milk of transgenic mice affects the growth of neonates.

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35 +/- 0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 +/- 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 +/- 0.33 g) and nontransgenic (0.91 +/- 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.

Mammary expression of new genes to combat mastitis.

Continual advances in the ability to produce transgenic animals make it likely that such animals will become important components of animal agriculture. The full benefit of the technology, and justification of its initial cost outlay, will be dependent on the establishment within these animals of new traits not easily achievable by other means. Potential applications include enhanced nutrient digestibility with reduced fecal losses, significantly altered milk composition with superior nutritional properties, and enhanced disease resistance. Our goal is to enhance mastitis resistance of dairy cows by enabling the cells of the mammary gland to secrete additional antibacterial proteins. Proof of concept has been obtained through experimentation with a transgenic mouse model. Three lines of mice were developed that produce varying levels of lysostaphin in their milk. This protein has potent anti-staphylococcal activity and its secretion into milk confers substantial resistance to infection caused by intramammary challenge with Staphylococcus aureus, a major mastitis pathogen. Additional antibacterial proteins are being sought that will complement lysostaphin. A potential benefit of transgenic application of antibacterial proteins is the concomitant sparing in the agricultural use of antibiotics currently used as human therapeutics. Antibacterial proteins, such as lysostaphin, are not typically used as injectable or oral therapeutics because of immune-mediated or digestive destruction of their activity. In contrast, the immune system of transgenic animals will not consider the transgenic protein as being foreign. In addition we are exploring the potential of involution or mastitis responsive promoter elements for use in subsequent transgenic experiments designed to restrict lysostaphin production to these important time points. It is anticipated that genomics will play a role in unveiling candidate genes whose promoter elements will enable desired temporal expression patterns. The transgenic approach to insertion of new genetic material into agriculturally important animals is feasible but requires extensive prior evaluation of the transgene and transgene product in model systems.

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Application of lysostaphin-producing lactobacilli to control staphylococcal food poisoning in meat products.

The potential of lysostaphin-producing strains of Lactobacillus curvatus (Lys+) to prevent food-borne illness by Staphylococcus aureus was investigated under practical conditions. A response surface model was developed to estimate the effect of pH, temperature, and salt concentration on the lysostaphin activity. The model was applied to fermenting sausages, and a 90% reduction of lysostaphin activity at ripening was predicted. The residual was sufficiently high to reduce staphylococcal counts by 10(4) to 10(5) CFU/g within 2 to 3 days to below the level of detection. These results were obtained in pilot scale experiments with L. curvatus (Lys+) as a starter culture and S. aureus as well as Staphylococcus carnosus as model contaminants. The applicability of L. curvatus (Lys+) as a protective culture was studied in a mayonnaise-based meat salad. Upon incubation at 25 degrees C the staphylococci were rapidly killed within 24 h, whereas in the presence of the isogenic Lys(-) strain the staphylococci grew up to numbers of 10(7) CFU/g.

Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.

The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.

Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei.

The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102S. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.

Antagonistic activities of coagulase-positive staphylococci.

Antagonistic activities were investigated by Frédérioq's plate method in 1,014 coagulase-positive staphylococcal strains of different species and origins. Staphylococcin effect was demonstrated in 12 (2%) of the 559 Staphylococcus aureus strains, in 51 (18%) of the 283 S. intermedius strains, and in 1 (3%) of the 36 S. hyicus subsp. chromogenes strains. Lysostaphin was identified in the 15 (5%) S. intermedius strains. In addition, heat-stable bacteriostatic agent was detected in 45 (33%) and heat-labile bacteriolytic agent in 7 (5%) of the 136 S. hyicus subsp. hyicus strains. An attempt was made at antagonistic activity typing in all of the active staphylococcal strains.

Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus.

Mutants of Staphylococcus staphylolyticus incapable of producing an extracellular staphylolytic glycylglycine endopeptidase were isolated and found to have cells in the population susceptible to lysis by this enzyme, as did the wild-type organism under conditions in which the endopeptidase was not produced. These results suggest that cultures of this organism normally contain a heterogeneous population of cells with regard to cell wall composition and susceptibility to the enzyme. Production of the endopeptidase appears to act as a selective pressure which removes the susceptible cells in the population as the enzyme appears in the medium. A comparison of the peptidoglycan of the wild-type organism grown under conditions in which the endopeptidase was produced with that of this organism grown under nonproducing conditions and with those of endopeptidase-less mutants showed that in the presence of the endopeptidase the cell population had peptidoglycan with shorter peptide cross bridges and a greater percentage of serine in these cross bridges than was found in cells grown in the absence of the enzyme. The inability of the endopeptidase to hydrolyze glycylserine and serylglycine peptide bonds suggests that at least part of the resistance this organism has to the endopeptidase is due to relative amounts of serine found in the peptide cross bridges of some cells in the population.