Limits...
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.

Show MeSH

Related in: MedlinePlus

MAG_5040 western blotting reactivity with sera collected from naturally infected hosts, and indirect detection of homologs in selected mycoplasma species.200 ng of cleaved rMAG_5040 were run in single well in 10% polyacrylamide gels and probed with sera collected alternatively from Piedmont goats naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) with rabbit hyperimmune sera raised against M. agalactiae PG2T (1), M. mycoides subsp. capri PG3 (2), M. capricolum subsp. capricolum CK (3), M. arginini G230 (4), M. canadense C275 (5), M. ovipneumoniae Y98 (6), M. putrefaciens KS1 (7), M. mycoides subsp. capri LC (8), and M. capricolum subsp. capripneumoniae (9). Lane 10 equals lane 1 except that no primary antibody was used.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585158&req=5

pone-0057775-g006: MAG_5040 western blotting reactivity with sera collected from naturally infected hosts, and indirect detection of homologs in selected mycoplasma species.200 ng of cleaved rMAG_5040 were run in single well in 10% polyacrylamide gels and probed with sera collected alternatively from Piedmont goats naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) with rabbit hyperimmune sera raised against M. agalactiae PG2T (1), M. mycoides subsp. capri PG3 (2), M. capricolum subsp. capricolum CK (3), M. arginini G230 (4), M. canadense C275 (5), M. ovipneumoniae Y98 (6), M. putrefaciens KS1 (7), M. mycoides subsp. capri LC (8), and M. capricolum subsp. capripneumoniae (9). Lane 10 equals lane 1 except that no primary antibody was used.

Mentions: In order to select a number of negative sheep from a flock with no history of CA, 10 sheep (group A) where tested by mycoplasma isolation, rP48 ELISA, and western blotting (see Material and Methods). Five group A sheep negative both serologically and culturally were moved to a mycoplasma-infected flock (group B). When rMAG_5040 was probed with sera collected from group A sheep placed in close contact with group B sheep, antibodies against MAG_5040 could be detected starting from T1 (2 weeks later) up to T18 (36 weeks, 9 months) in 3 out of 5 sheep. Western blotting evaluation performed on total protein lysates of M. agalactiae PG2T against the same sera showed that the 3 group A sheep were already infected at T1. Mycoplasma isolation and rP48 ELISA were consistent with western blotting. Sera collected during the same sampling occasions from culturally and serologically negative sheep never reacted with rMAG_5040 (data not shown). Figure 5B summarizes the results of the longitudinal study. A strong reactivity was also observed when rMAG_5040 was tested against the two panels of well characterized sera obtained from CA outbreaks occurred in Piedmont goats and Sicilian sheep (Figure 6A and 6B).


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 western blotting reactivity with sera collected from naturally infected hosts, and indirect detection of homologs in selected mycoplasma species.200 ng of cleaved rMAG_5040 were run in single well in 10% polyacrylamide gels and probed with sera collected alternatively from Piedmont goats naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) with rabbit hyperimmune sera raised against M. agalactiae PG2T (1), M. mycoides subsp. capri PG3 (2), M. capricolum subsp. capricolum CK (3), M. arginini G230 (4), M. canadense C275 (5), M. ovipneumoniae Y98 (6), M. putrefaciens KS1 (7), M. mycoides subsp. capri LC (8), and M. capricolum subsp. capripneumoniae (9). Lane 10 equals lane 1 except that no primary antibody was used.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057775-g006: MAG_5040 western blotting reactivity with sera collected from naturally infected hosts, and indirect detection of homologs in selected mycoplasma species.200 ng of cleaved rMAG_5040 were run in single well in 10% polyacrylamide gels and probed with sera collected alternatively from Piedmont goats naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) with rabbit hyperimmune sera raised against M. agalactiae PG2T (1), M. mycoides subsp. capri PG3 (2), M. capricolum subsp. capricolum CK (3), M. arginini G230 (4), M. canadense C275 (5), M. ovipneumoniae Y98 (6), M. putrefaciens KS1 (7), M. mycoides subsp. capri LC (8), and M. capricolum subsp. capripneumoniae (9). Lane 10 equals lane 1 except that no primary antibody was used.
Mentions: In order to select a number of negative sheep from a flock with no history of CA, 10 sheep (group A) where tested by mycoplasma isolation, rP48 ELISA, and western blotting (see Material and Methods). Five group A sheep negative both serologically and culturally were moved to a mycoplasma-infected flock (group B). When rMAG_5040 was probed with sera collected from group A sheep placed in close contact with group B sheep, antibodies against MAG_5040 could be detected starting from T1 (2 weeks later) up to T18 (36 weeks, 9 months) in 3 out of 5 sheep. Western blotting evaluation performed on total protein lysates of M. agalactiae PG2T against the same sera showed that the 3 group A sheep were already infected at T1. Mycoplasma isolation and rP48 ELISA were consistent with western blotting. Sera collected during the same sampling occasions from culturally and serologically negative sheep never reacted with rMAG_5040 (data not shown). Figure 5B summarizes the results of the longitudinal study. A strong reactivity was also observed when rMAG_5040 was tested against the two panels of well characterized sera obtained from CA outbreaks occurred in Piedmont goats and Sicilian sheep (Figure 6A and 6B).

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