Dataset Information

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

ABSTRACT: The survival, replication, and virulence of mycoplasmas depend on their ability to capture and import host-derived nutrients using poorly characterized membrane proteins. Previous studies on the important bovine pathogen Mycoplasma bovis demonstrated that the amino-terminal end of an immunogenic 226-kDa (P226) protein, encoded by milA (the full-length product of which has a predicted molecular weight of 303 kDa), had lipase activity. The predicted sequence of MilA contains glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. We mutagenized the gene to facilitate expression of a series of regions spanning the gene in Escherichia coli Using monospecific antibodies against these recombinant proteins, we showed that MilA was proteolytically processed into 226-kDa and 50-kDa fragments that were both partitioned into the detergent phase by Triton X-114 phase fractionation. Trypsin treatment of intact cells showed that P226 was surface exposed. In vitro, the recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and to a variety of lipids. The MilA fragments were also shown to bind heparin. Antibody against the carboxyl-terminal fragment inhibited the growth of M. bovisin vitro This carboxyl end also bound and hydrolyzed ATP, suggestive of a potential role as an autotransporter. Our studies have demonstrated that DUF445 has lipid binding activity and that MilA is a multifunctional protein that may play multiple roles in the pathogenesis of infection with M. bovis.

SUBMITTER: Adamu JY

PROVIDER: S-EPMC7240078 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

Adamu James Yazah JY Wawegama Nadeeka Kumari NK Kanci Condello Anna A Marenda Marc Serge MS Markham Philip Francis PF Browning Glenn Francis GF Tivendale Kelly Anne KA

Infection and immunity 20200520 6

The survival, replication, and virulence of mycoplasmas depend on their ability to capture and import host-derived nutrients using poorly characterized membrane proteins. Previous studies on the important bovine pathogen <i>Mycoplasma bovis</i> demonstrated that the amino-terminal end of an immunogenic 226-kDa (P226) protein, encoded by <i>milA</i> (the full-length product of which has a predicted molecular weight of 303 kDa), had lipase activity. The predicted sequence of MilA contains glycosam ...[more]

PMID: 32253247

Similar Datasets

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:Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210-269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αβ-crystallin knockout cell line BoMac-cryab-, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab- induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.

Project description:Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here, we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of mycoplasma metabolite extracts revealed significant differences in the steady-state levels of many metabolites in central carbon metabolism, while 13C stable isotope labeling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilization, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterizing significant differences in the metabolism of different bacterial species and in improving genome annotation. IMPORTANCE Mycoplasmas are pathogenic bacteria that cause serious chronic infections in production animals, resulting in considerable losses worldwide, as well as causing disease in humans. These bacteria have extremely reduced genomes and are thought to have limited metabolic flexibility, even though they are highly successful persistent parasites in a diverse number of species. The extent to which different Mycoplasma species are capable of catabolizing host carbon sources and nutrients, or synthesizing essential metabolites, remains poorly defined. We have used advanced metabolomic techniques to identify metabolic pathways that are active in two species of Mycoplasma that infect distinct hosts (poultry and cattle). We show that these species exhibit marked differences in metabolite steady-state levels and carbon source utilization. This information has been used to functionally characterize previously unknown genes in the genomes of these pathogens. These species-specific differences are likely to reflect important differences in host nutrient levels and pathogenic mechanisms.

Dataset Information

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

Publications

Mycoplasma bovis Membrane Protein MilA Is a Multifunctional Lipase with Novel Lipid and Glycosaminoglycan Binding Activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets