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

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.

Figure 1. 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 PG2^T proteins are indicated above each row. Phylogeny based on the amino acid alignment of the SNc homologs is shown on the left.

Figure 2. Multiple sequence alignment of the TNASE_3 domain of M. agalactiae PG2^T MAG_5040 with homologous proteins identified in other mycoplasma species, and with the SNc of Staphylococcus aures.

Positions are numbered according to the first amino acid of this domain in each bacterial species. Conserved amino acid residues involved in cations binding and encompassing the catalytic site are shown in boldface. An additionally conserved glycine residue is underlined and in boldface. Asterisks indicate positions conserved in all the bacteria aligned.

Figure 3. 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.

Figure 4. Effects of Ca²⁺, Mg²⁺, combined Ca²⁺+Mg²⁺, ionic strength, and temperature on MAG_5040 nuclease activity.

The nuclease activity and stability under different tested conditions were evaluated by loading on a 1% agarose gel approximately 10 µl of each of the endpoint reactions. The far left lane of each panel was loaded with the molecular weight marker (MW). Both 1 Kb (left and central panels) and 1 Kb Plus (top and bottom right panels) DNA ladders were used (Invitrogen). In each panel, lanes designated C were loaded with untreated plasmid DNA in agarose loading buffer. Concentrations expressed in mM and temperatures in °C are indicated in the appropriate panels (Ca²⁺, top left panel; Mg²⁺, top middle panel; combined Ca²⁺+Mg²⁺, top right panel; ionic strength, bottom left and middle panels; and temperature, bottom right panel).

Figure 5. 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.

Figure 6. 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 PG2^T (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.