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Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

ABSTRACT: This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca(2+) at 22-65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-?B p65 and Bax, and inhibition of I??? and Bcl-2. In contrast, rMbovNase(?181-342) without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.

SUBMITTER: Zhang H

PROVIDER: S-EPMC4881454 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

Zhang Hui H Zhao Gang G Guo Yusi Y Menghwar Harish H Chen Yingyu Y Chen Huanchun H Guo Aizhen A

International journal of molecular sciences 20160429 5

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca(2+)-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed ...[more]

PMID: 27136546

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Project description:UnlabelledAlthough the complete genome sequences of three strains of Mycoplasma bovis are available, few studies have examined gene function in this important pathogen. Mycoplasmas lack the biosynthetic machinery for the de novo synthesis of nucleic acid precursors, so nucleases are likely to be essential for them to acquire nucleotide precursors. Three putative membrane nucleases have been annotated in the genome of M. bovis strain PG45, MBOVPG45_0089 and MBOVPG45_0310, both of which have the thermonuclease (TNASE_3) functional domain, and MBOVPG45_0215 (mnuA), which has an exonuclease/endonuclease/phosphatase domain. While previous studies have demonstrated the function of TNASE_3 domain nucleases in several mycoplasmas, quantitative comparisons of the contributions of different nucleases to cellular nuclease activity have been lacking. Mapping of a library of 319 transposon mutants of M. bovis PG45 by direct genome sequencing identified mutants with insertions in MBOVPG45_0310 (the Δ0310 mutant) and MBOVPG45_0215 (the Δ0215 mutant). In this study, the detection of the product of MBOVPG45_0215 in the Triton X-114 fraction of M. bovis cell lysates, its cell surface exposure, and its predicted signal peptide suggested that it is a surface-exposed lipoprotein nuclease. Comparison of a ΔmnuA mutant with wild-type M. bovis on native and denatured DNA gels and in digestion assays using double-stranded phage λ DNA and closed circular plasmid DNA demonstrated that inactivation of this gene abolishes most of the cellular exonuclease and endonuclease activity of M. bovis. This activity could be fully restored by complementation with the wild-type mnuA gene, demonstrating that MnuA is the major cellular nuclease of M. bovis.ImportanceNucleases are thought to be important contributors to virulence and crucial for the maintenance of a nutritional supply of nucleotides in mycoplasmas that are pathogenic in animals. This study demonstrates for the first time that of the three annotated cell surface nuclease genes in an important pathogenic mycoplasma, the homologue of the thermostable nuclease identified in Gram-positive bacteria is responsible for the majority of the nuclease activity detectable in vitro.

Project description:Microbial access to host nutrients is a key factor of the host-pathogen interplay. With their nearly minimal genome, wall-less bacteria of the class Mollicutes have limited metabolic capacities and largely depend on host nutrients for their survival. Despite these limitations, host-restricted mycoplasmas are widely distributed in nature and many species are pathogenic for humans and animals. Yet, only partial information is available regarding the mechanisms evolved by these minimal pathogens to meet their nutrients and the contribution of these mechanisms to virulence. By using the ruminant pathogen Mycoplasma bovis as a model system, extracellular DNA (eDNA) was identified as a limiting nutrient for mycoplasma proliferation under cell culture conditions. Remarkably, the growth-promoting effect induced by supplementation with eDNA was associated with important cytotoxicity for actively dividing host cells, but not confluent monolayers. To identify biological functions mediating M. bovis cytotoxicity, we produced a library of transposon knockout mutants and identified three critical genomic regions whose disruption was associated with a non-cytopathic phenotype. The coding sequences (CDS) disrupted in these regions pointed towards pyruvate metabolism as contributing to M. bovis cytotoxicity. Hydrogen peroxide was found responsible for eDNA-mediated M. bovis cytotoxicity, and non-cytopathic mutants were unable to produce this toxic metabolic compound. In our experimental conditions, no contact between M. bovis and host cells was required for cytotoxicity. Further analyses revealed important intra-species differences in eDNA-mediated cytotoxicity and H2O2 production, with some strains displaying a cytopathic phenotype despite no H2O2 production. Interestingly, the genome of strains PG45 and HB0801 were characterized by the occurrence of insertion sequences (IS) at close proximity to several CDSs found disrupted in non-cytopathic mutants. Since PG45 and HB0801 produced no or limited amount of H2O2, IS-elements might influence H2O2 production in M. bovis. These results confirm the multifaceted role of eDNA in microbial communities and further identify this ubiquitous material as a nutritional trigger of M. bovis cytotoxicity. M. bovis may thus take advantage of the multiple sources of eDNA in vivo to modulate its interaction with host cells, a way for this minimal pathogen to overcome its limited coding capacity.

Dataset Information

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

Publications

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity.

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