Expression of Active Monomeric and Dimeric Nuclease A from the Gram-Positive Streptococcus gordonii Surface Protein Expression System

Dutton, Emma K.; Ottum, Sean A.; Bolken, Tove’ C.; Franke, Christine A.; Hruby, Dennis E.

doi:10.1006/prep.2000.1223

Cited by 9 publications

(7 citation statements)

References 38 publications

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“…The portion of spd1 encoding the mature protein was cloned into the Streptococcus gordonii expression vector pSMB104/ pSTOP (kindly provided by Siga Technologies, Corvalis, Oreg.). The protocol outlined by Dutton et al was then followed for transformation and expression of the molecule in S. gordonii strain GP251 (7).…”

Section: Methodsmentioning

confidence: 99%

The In Vitro Interaction ofStreptococcus pyogeneswith Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase

Broudy

Pancholi²,

Fischetti

2002

Infect Immun

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The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harbors speC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage.

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Section: Methodsmentioning

confidence: 99%

The In Vitro Interaction ofStreptococcus pyogeneswith Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase

Broudy

Pancholi²,

Fischetti

2002

Infect Immun

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“…The initial phase of digestion is characterized as predominantly endonucleolytic, with only slight site preferences, followed by exonucleolytic degradation (17). Apart from its natural host, the staphylococcal nuclease was expressed in an enzymatic active form in a diverse spectrum of gram-positive and gram-negative bacterial species (5,8,23,31).…”

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confidence: 99%

Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease

Haidinger

Mayr

Szostak³

et al. 2003

Appl Environ Microbiol

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The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage X174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.Controlled expression of the cloned X174 lysis gene E results in the formation of empty bacterial cell envelopes (ghosts) (28, 33). Applicable in a broad range of gram-negative bacterial species, the protein E-mediated lysis procedure gives rise to a new class of genetically inactivated candidate vaccines (11,16).In contrast to the majority of tested bacterial species, which are completely inactivated by the lysis process (9, 16), the production of bacterial ghosts from Escherichia coli K-12 species is accompanied by the rare detection of nonlysed inactivated or reproductive cells within the ghost preparation. In previous studies, when green fluorescent protein-derived fluorescence was used for microscopic discrimination between ghosts and nonlysed cells, the percentage of nonlysed reproductive and inactivated E. coli cells was determined as 1.3% and 1.2%, respectively (13). The population of nonlysed cells was cytometrically subdivided into polarized and depolarized cells, and the detected populations were successfully quantified during the time course of lysis (14). The minimum ratios of nonlysed cells within the ghost preparations were cytometrically ascertained to be 4% for the polarized and 1% for the depolarized population, which correlated well with the results derived from classical microbiological procedures, indicating a minimum of 1% reproductive bacteri...

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“…SNUC is fully dependent on Ca 2ϩ , and supplementation with Mg 2ϩ has a stimulatory effect on DNase activity (8). Apart from its natural host, the staphylococcal nuclease has been expressed in its active form in various gram-positive and gram-negative bacteria (4,10,30,47). Recently, the E-mediated lysis and SNUC inactivation of E. coli K-12 strain NM522 has been described in detail (17), and in this study, the method was adapted for the first time to an EHEC strain at the fermentor scale.…”

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confidence: 99%

Bacterial Ghosts as an Oral Vaccine: a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge

et al. 2005

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Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several lifethreatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

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Expression of Active Monomeric and Dimeric Nuclease A from the Gram-Positive Streptococcus gordonii Surface Protein Expression System

Cited by 9 publications

References 38 publications

The In Vitro Interaction ofStreptococcus pyogeneswith Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase

The In Vitro Interaction ofStreptococcus pyogeneswith Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase

Escherichia coli Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease

Bacterial Ghosts as an Oral Vaccine: a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge

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