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Mycoplasma agalactiae MAG_5040 is a Mg2+-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection.

Cacciotto C, Addis MF, Coradduzza E, Carcangiu L, Nuvoli AM, Tore G, Dore GM, Pagnozzi D, Uzzau S, Chessa B, Pittau M, Alberti A - PLoS ONE (2013)

Bottom Line: In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search.In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection.The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy.

ABSTRACT
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

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Nuclease activity and substrate specificity of recombinant GST-MAG_5040.Approximately 4 µg of rGST-MAG_5040 were incubated with calf thymus dsDNA (top left panel), closed circular plasmid DNA (pGEX-2T/rMAG_5040, top right panel), phage M13 ssDNA (bottom left panel), or E. coli total RNA (bottom right panel). An aliquot of each reaction mixture was analysed at different times, as indicated for each lane. C indicates endpoint reaction of the negative undigested control (GST only). Both 1 Kb (upper panels) and 1 Kb Plus (bottom panels) DNA ladders were used (Invitrogen) and indicated as MW in far left lanes of each panel.
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pone-0057775-g003: Nuclease activity and substrate specificity of recombinant GST-MAG_5040.Approximately 4 µg of rGST-MAG_5040 were incubated with calf thymus dsDNA (top left panel), closed circular plasmid DNA (pGEX-2T/rMAG_5040, top right panel), phage M13 ssDNA (bottom left panel), or E. coli total RNA (bottom right panel). An aliquot of each reaction mixture was analysed at different times, as indicated for each lane. C indicates endpoint reaction of the negative undigested control (GST only). Both 1 Kb (upper panels) and 1 Kb Plus (bottom panels) DNA ladders were used (Invitrogen) and indicated as MW in far left lanes of each panel.

Mentions: In order to evaluate MAG_5040 nuclease activity, rGST-MAG_5040 was incubated with different nucleic acid substrates under different conditions. Since it has been demonstrated that most Mycoplasma nucleases show their maximum activity when Ca2+ and Mg2+ ions are combined in the reaction buffer [10], substrate specificity of MAG_5040 was initially evaluated in the presence of both 5 mM CaCl2 and 5 mM MgCl2. When linear dsDNA (calf thymus DNA) was incubated with GST-rMAG_5040, the amount of intact dsDNA decreased during incubation, and that was associated with the appearance of a smear in the gel, whose average size became lower over time (Figure 3, upper left panel), indicating an exonuclease activity of the protein. Similarly, endonuclease activity was assessed by using a dsDNA circular plasmid (pGEX-2T/rMAG_5040) as a substrate (Figure 3, upper right panel). Recombinant GST-MAG_5040 appeared to be far more active against ssDNA (M13 DNA) than against dsDNA. Indeed, high degree of ssDNA degradation could be instantly observed at T0. After 1 minute, M13 DNA was almost totally digested (Figure 3, lower left panel). Recombinant GST-MAG_5040 was also found to be active, although to a lesser extent, against total RNA extracted from E. coli (Figure 3, lower right panel). Digestions of the different substrate were always coupled with adequately matched negative controls (Figure 3). Optimal biochemical conditions were investigated and defined by evaluating the degree of degradation of plasmid DNA in the presence of increasing concentrations of divalent cations (Ca2+ and Mg2+), and by varying ionic strength (Na+ and K+), and temperature (Figure 4). Recombinant GST-MAG_5040 did not show any nuclease activity when reaction buffers were supplemented with EDTA (5 mM final concentration), confirming the biochemical requirement for divalent ions (data not shown). When reaction buffer was supplemented with increasing concentrations of MgCl2, a corresponding increase of the ability to digest circular dsDNA was also observed, with apparent maximum efficiency of rGST-MAG_5040 in the presence of 20 mM MgCl2 (Figure 4, top middle panel). On the contrary when MgCl2 was replaced with CaCl2 in similar experiments, a progressive inhibitory action of Ca2+ on the activity of rGST-MAG_5040 could be surprisingly observed (Figure 4, top left panel). Thus, with dsDNA as substrate, MAG_5040 shows a strong preference for Mg2+ over calcium ions. The efficiencies of dsDNA digestions conducted by combining 20 mM MgCl2 to 0.1 mM CaCl2 and by using 20 mM MgCl2 alone were comparable (Figure 4, top right panel).


Mycoplasma agalactiae MAG_5040 is a Mg2+-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection.

Cacciotto C, Addis MF, Coradduzza E, Carcangiu L, Nuvoli AM, Tore G, Dore GM, Pagnozzi D, Uzzau S, Chessa B, Pittau M, Alberti A - PLoS ONE (2013)

Nuclease activity and substrate specificity of recombinant GST-MAG_5040.Approximately 4 µg of rGST-MAG_5040 were incubated with calf thymus dsDNA (top left panel), closed circular plasmid DNA (pGEX-2T/rMAG_5040, top right panel), phage M13 ssDNA (bottom left panel), or E. coli total RNA (bottom right panel). An aliquot of each reaction mixture was analysed at different times, as indicated for each lane. C indicates endpoint reaction of the negative undigested control (GST only). Both 1 Kb (upper panels) and 1 Kb Plus (bottom panels) DNA ladders were used (Invitrogen) and indicated as MW in far left lanes of each panel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585158&req=5

pone-0057775-g003: Nuclease activity and substrate specificity of recombinant GST-MAG_5040.Approximately 4 µg of rGST-MAG_5040 were incubated with calf thymus dsDNA (top left panel), closed circular plasmid DNA (pGEX-2T/rMAG_5040, top right panel), phage M13 ssDNA (bottom left panel), or E. coli total RNA (bottom right panel). An aliquot of each reaction mixture was analysed at different times, as indicated for each lane. C indicates endpoint reaction of the negative undigested control (GST only). Both 1 Kb (upper panels) and 1 Kb Plus (bottom panels) DNA ladders were used (Invitrogen) and indicated as MW in far left lanes of each panel.
Mentions: In order to evaluate MAG_5040 nuclease activity, rGST-MAG_5040 was incubated with different nucleic acid substrates under different conditions. Since it has been demonstrated that most Mycoplasma nucleases show their maximum activity when Ca2+ and Mg2+ ions are combined in the reaction buffer [10], substrate specificity of MAG_5040 was initially evaluated in the presence of both 5 mM CaCl2 and 5 mM MgCl2. When linear dsDNA (calf thymus DNA) was incubated with GST-rMAG_5040, the amount of intact dsDNA decreased during incubation, and that was associated with the appearance of a smear in the gel, whose average size became lower over time (Figure 3, upper left panel), indicating an exonuclease activity of the protein. Similarly, endonuclease activity was assessed by using a dsDNA circular plasmid (pGEX-2T/rMAG_5040) as a substrate (Figure 3, upper right panel). Recombinant GST-MAG_5040 appeared to be far more active against ssDNA (M13 DNA) than against dsDNA. Indeed, high degree of ssDNA degradation could be instantly observed at T0. After 1 minute, M13 DNA was almost totally digested (Figure 3, lower left panel). Recombinant GST-MAG_5040 was also found to be active, although to a lesser extent, against total RNA extracted from E. coli (Figure 3, lower right panel). Digestions of the different substrate were always coupled with adequately matched negative controls (Figure 3). Optimal biochemical conditions were investigated and defined by evaluating the degree of degradation of plasmid DNA in the presence of increasing concentrations of divalent cations (Ca2+ and Mg2+), and by varying ionic strength (Na+ and K+), and temperature (Figure 4). Recombinant GST-MAG_5040 did not show any nuclease activity when reaction buffers were supplemented with EDTA (5 mM final concentration), confirming the biochemical requirement for divalent ions (data not shown). When reaction buffer was supplemented with increasing concentrations of MgCl2, a corresponding increase of the ability to digest circular dsDNA was also observed, with apparent maximum efficiency of rGST-MAG_5040 in the presence of 20 mM MgCl2 (Figure 4, top middle panel). On the contrary when MgCl2 was replaced with CaCl2 in similar experiments, a progressive inhibitory action of Ca2+ on the activity of rGST-MAG_5040 could be surprisingly observed (Figure 4, top left panel). Thus, with dsDNA as substrate, MAG_5040 shows a strong preference for Mg2+ over calcium ions. The efficiencies of dsDNA digestions conducted by combining 20 mM MgCl2 to 0.1 mM CaCl2 and by using 20 mM MgCl2 alone were comparable (Figure 4, top right panel).

Bottom Line: In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search.In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection.The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, Italy.

ABSTRACT
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

Show MeSH
Related in: MedlinePlus