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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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Related in: MedlinePlus

MAG_5040 antigenic properties.(A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10% polyacrylamide gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes designated MW.
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pone-0057775-g005: MAG_5040 antigenic properties.(A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10% polyacrylamide gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes designated MW.

Mentions: A previous study suggested that MAG_5040 is a surface lipoprotein expressed in cultured M. agalactiae PG2T[15]. In order to investigate the antigenic properties of MAG_5040, and to evaluate its expression in naturally infected hosts, a longitudinal study was performed on sera collected from sheep at different stages of infection. In addition, the reactivity of a panel of well-characterized goat and sheep sera from two unrelated outbreaks was also evaluated. At first, the antigenicity of MAG_5040 was established, and the sensitivity of western blotting based on its recombinant form was then assessed. Figure 5A shows the reactivity of a pool of sera obtained from naturally infected sheep against decreasing amounts of the cleaved recombinant protein. A strong reactivity of the pool of sera against rMAG_5040 could be observed down to 12 ng of recombinant protein, with 6 ng of protein still associating to a weak signal. A pool of sera obtained from negative sheep tested always negative in similar experiments (data not shown).


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)

MAG_5040 antigenic properties.(A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10% polyacrylamide gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes designated MW.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585158&req=5

pone-0057775-g005: MAG_5040 antigenic properties.(A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10% polyacrylamide gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes designated MW.
Mentions: A previous study suggested that MAG_5040 is a surface lipoprotein expressed in cultured M. agalactiae PG2T[15]. In order to investigate the antigenic properties of MAG_5040, and to evaluate its expression in naturally infected hosts, a longitudinal study was performed on sera collected from sheep at different stages of infection. In addition, the reactivity of a panel of well-characterized goat and sheep sera from two unrelated outbreaks was also evaluated. At first, the antigenicity of MAG_5040 was established, and the sensitivity of western blotting based on its recombinant form was then assessed. Figure 5A shows the reactivity of a pool of sera obtained from naturally infected sheep against decreasing amounts of the cleaved recombinant protein. A strong reactivity of the pool of sera against rMAG_5040 could be observed down to 12 ng of recombinant protein, with 6 ng of protein still associating to a weak signal. A pool of sera obtained from negative sheep tested always negative in similar experiments (data not shown).

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