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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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MAG_5040 organization and position of homologs in the genome of phylogenetically related bacteria.(A) MAG_5040 schematic diagram. MAG_5040 is comprised of 390 amino acids and contains a hydrophobic N-terminal signal sequence and a prokaryotic lipoprotein cleavage site (indicated by the grey box). Amino acids 182 through 343 have significant identity and similarity with the TNASE_3 domain profile of the Staphylococcus aureus thermonuclease (SNc). Conserved amino acid residues involved in binding of divalent ions are indicated by arrowed vertical lines. Position of amino acids comprising the active catalytic site are shown as vertical lines with round tips. (B) Physical map of mycoplasma homologs of the putative MAG_5030-MAG_5040-MAG_5050-MAG_5060-MAG_5070 ABC transport system. Homologous proteins are shaded in the same pattern, and their amino acid identities to the corresponding M. agalactiae PG2T proteins are indicated above each row. Phylogeny based on the amino acid alignment of the SNc homologs is shown on the left.
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pone-0057775-g001: MAG_5040 organization and position of homologs in the genome of phylogenetically related bacteria.(A) MAG_5040 schematic diagram. MAG_5040 is comprised of 390 amino acids and contains a hydrophobic N-terminal signal sequence and a prokaryotic lipoprotein cleavage site (indicated by the grey box). Amino acids 182 through 343 have significant identity and similarity with the TNASE_3 domain profile of the Staphylococcus aureus thermonuclease (SNc). Conserved amino acid residues involved in binding of divalent ions are indicated by arrowed vertical lines. Position of amino acids comprising the active catalytic site are shown as vertical lines with round tips. (B) Physical map of mycoplasma homologs of the putative MAG_5030-MAG_5040-MAG_5050-MAG_5060-MAG_5070 ABC transport system. Homologous proteins are shaded in the same pattern, and their amino acid identities to the corresponding M. agalactiae PG2T proteins are indicated above each row. Phylogeny based on the amino acid alignment of the SNc homologs is shown on the left.

Mentions: The MAG_5040 gene encodes a putative protein of 390 amino acid residues with a predicted molecular mass of 44.67 kDa and a pI of about 8.5. LipoP analysis classifies MAG_5040 as a putative lipoprotein, with a classical signal peptide of 25 amino acids and a typical cysteine cleavage site at residue 25 (Figure 1A). PROSITE scan of MAG_5040 reveals the presence of the nuclease domain TNASE_3, characteristic of the S. aureus thermonuclease [16], [17], [18], [19]. The TNASE_3 domain (Figure 1A, Figure 2) spans positions 182–343, and contains two aspartates and a threonine located at positions 195, 225, and 226, respectively. These amino acids are conserved among mycoplasmas of the M. hominis group, and are involved in the binding of divalent ions. The amino acid residues arginine, glutamate, and arginine, respectively located in the TNASE_3 motive at positions 220, 228, and 273 are also strictly conserved and comprise the active catalytic site. Additionally, a glycine residue is conserved at position 296. Figure 1B shows the alignment of MAG_5040 gene and its flanking regions with the corresponding regions of 10 selected Mycoplasma species and with S. aureus subspecies aureus. In M. agalactiae, MAG_5040 is located immediately upstream of 3 genes encoding an ABC transport system. MAG_5050, the predicted ATP-binding protein, contains the two ATP-binding domains that couple ATP hydrolysis to transport. The two genes MAG_5060 and MAG_5070 encode the transmembrane permeases that are associated with the import of the substrate. MAG_5050, MAG_5060, MAG_5070 shows similarity to putative sugar ABC transporter components of mycoplasma species belonging to the hominis group (Figure 1B), and of other distant bacteria. Indeed BLASTP sequence comparisons identify homologues of MAG_5050, MAG_5060, MAG_5070 in M. crocodyli, M. pneumoniae, M. gallisepticum, Achromobacter spp., Roseobacter sp., Clostridium sp., Octadecabacter antarcticus, Lactobacillus spp., and many other bacterial genera (data not shown). Similarly, MAG_5040 homologues are present in about 60 Gram-negative and Gram-positive bacterial species. With one exception (M. synoviae, Figure 1B), the sequential distribution of MAG_5040 and of the ABC transporter genes is highly conserved in mycoplasmas belonging to the M. hominis group (Figure 1B). In addition, position of MAG_5030 upstream of the putative SNase is also conserved, at least in the same mycoplasmas. Mycoplasma agalactiae MAG_5030 encodes the antigenic lipoprotein P80 [31]. On the contrary MAG_5080, located downstream of the ABC transporter, shows significant homology uniquely with the MBOVPG45_0305 gene of M. bovis, also positioned in this mycoplasma downstream of the ABC transporter permeases, but with the interposition of the small open reading frame MBOVPG45_0306 (Figure 1B).


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 organization and position of homologs in the genome of phylogenetically related bacteria.(A) MAG_5040 schematic diagram. MAG_5040 is comprised of 390 amino acids and contains a hydrophobic N-terminal signal sequence and a prokaryotic lipoprotein cleavage site (indicated by the grey box). Amino acids 182 through 343 have significant identity and similarity with the TNASE_3 domain profile of the Staphylococcus aureus thermonuclease (SNc). Conserved amino acid residues involved in binding of divalent ions are indicated by arrowed vertical lines. Position of amino acids comprising the active catalytic site are shown as vertical lines with round tips. (B) Physical map of mycoplasma homologs of the putative MAG_5030-MAG_5040-MAG_5050-MAG_5060-MAG_5070 ABC transport system. Homologous proteins are shaded in the same pattern, and their amino acid identities to the corresponding M. agalactiae PG2T proteins are indicated above each row. Phylogeny based on the amino acid alignment of the SNc homologs is shown on the left.
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Related In: Results  -  Collection

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pone-0057775-g001: MAG_5040 organization and position of homologs in the genome of phylogenetically related bacteria.(A) MAG_5040 schematic diagram. MAG_5040 is comprised of 390 amino acids and contains a hydrophobic N-terminal signal sequence and a prokaryotic lipoprotein cleavage site (indicated by the grey box). Amino acids 182 through 343 have significant identity and similarity with the TNASE_3 domain profile of the Staphylococcus aureus thermonuclease (SNc). Conserved amino acid residues involved in binding of divalent ions are indicated by arrowed vertical lines. Position of amino acids comprising the active catalytic site are shown as vertical lines with round tips. (B) Physical map of mycoplasma homologs of the putative MAG_5030-MAG_5040-MAG_5050-MAG_5060-MAG_5070 ABC transport system. Homologous proteins are shaded in the same pattern, and their amino acid identities to the corresponding M. agalactiae PG2T proteins are indicated above each row. Phylogeny based on the amino acid alignment of the SNc homologs is shown on the left.
Mentions: The MAG_5040 gene encodes a putative protein of 390 amino acid residues with a predicted molecular mass of 44.67 kDa and a pI of about 8.5. LipoP analysis classifies MAG_5040 as a putative lipoprotein, with a classical signal peptide of 25 amino acids and a typical cysteine cleavage site at residue 25 (Figure 1A). PROSITE scan of MAG_5040 reveals the presence of the nuclease domain TNASE_3, characteristic of the S. aureus thermonuclease [16], [17], [18], [19]. The TNASE_3 domain (Figure 1A, Figure 2) spans positions 182–343, and contains two aspartates and a threonine located at positions 195, 225, and 226, respectively. These amino acids are conserved among mycoplasmas of the M. hominis group, and are involved in the binding of divalent ions. The amino acid residues arginine, glutamate, and arginine, respectively located in the TNASE_3 motive at positions 220, 228, and 273 are also strictly conserved and comprise the active catalytic site. Additionally, a glycine residue is conserved at position 296. Figure 1B shows the alignment of MAG_5040 gene and its flanking regions with the corresponding regions of 10 selected Mycoplasma species and with S. aureus subspecies aureus. In M. agalactiae, MAG_5040 is located immediately upstream of 3 genes encoding an ABC transport system. MAG_5050, the predicted ATP-binding protein, contains the two ATP-binding domains that couple ATP hydrolysis to transport. The two genes MAG_5060 and MAG_5070 encode the transmembrane permeases that are associated with the import of the substrate. MAG_5050, MAG_5060, MAG_5070 shows similarity to putative sugar ABC transporter components of mycoplasma species belonging to the hominis group (Figure 1B), and of other distant bacteria. Indeed BLASTP sequence comparisons identify homologues of MAG_5050, MAG_5060, MAG_5070 in M. crocodyli, M. pneumoniae, M. gallisepticum, Achromobacter spp., Roseobacter sp., Clostridium sp., Octadecabacter antarcticus, Lactobacillus spp., and many other bacterial genera (data not shown). Similarly, MAG_5040 homologues are present in about 60 Gram-negative and Gram-positive bacterial species. With one exception (M. synoviae, Figure 1B), the sequential distribution of MAG_5040 and of the ABC transporter genes is highly conserved in mycoplasmas belonging to the M. hominis group (Figure 1B). In addition, position of MAG_5030 upstream of the putative SNase is also conserved, at least in the same mycoplasmas. Mycoplasma agalactiae MAG_5030 encodes the antigenic lipoprotein P80 [31]. On the contrary MAG_5080, located downstream of the ABC transporter, shows significant homology uniquely with the MBOVPG45_0305 gene of M. bovis, also positioned in this mycoplasma downstream of the ABC transporter permeases, but with the interposition of the small open reading frame MBOVPG45_0306 (Figure 1B).

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