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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.
Highlights
Mycoplasmas are the smallest and simplest self-replicating prokaryotes
With one exception (M. synoviae, Figure 1B), the sequential distribution of MAG_5040 and of the ATP-binding cassette (ABC) transporter genes is highly conserved in mycoplasmas belonging to the M. hominis group (Figure 1B)
M. agalactiae is the etiological agent of contagious agalactia (CA), a serious disease of sheep and goats reported worldwide and endemic in most Mediterranean countries [32]
Summary
Mycoplasmas are the smallest and simplest self-replicating prokaryotes. They evolved from Gram-positive bacteria following a regressive process that led to the reduction of genomic resources to an essential minimum [1]. Mycoplasmas evolutionary constraints in host adaptation forced to maintain membrane key components for extracellular degradation of nucleic acids, such as nucleases, and for transporting selected nucleotides precursors through the membrane, such as ATP-binding cassette (ABC) transport systems [1]. We demonstrated the expression of MAG_5040, which was classified, according to gene ontology analyses, as a surface lipoprotein containing the conserved SNase domain of Staphylococcus aureus (S. aureus). In this study we characterized the in vitro activity of the putative M. agalactiae SNase MAG_5040, and we investigated its expression and antigenic properties during natural infection. Recombinant cleaved MAG_5040 was used to detect specific antibodies during different stages of infection in the natural hosts (sheep and goats), and to determine its reactivity with hyperimmune sera raised against selected mycoplasma species, as a preliminary investigation of potential SNase homologues expressed in other Mycoplasma species
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