Fig 1.
Schematic presentation M. bovis PG45 5′ nucleotidase and phylogenetic tree of its homologs.
(A) Schematic diagram of the surface-exposed M. bovis 5’-NT lipoprotein is depicted based on prediction from UniProtKB software at Swiss-Prot (https://www.uniprot.org/). The diagram highlights key features, including the coiled coil (gray) sequence, calcineurin-like phosphoesterase (Metallophos, PF00149; violet), and 5′-nucleotidase (5_nucleotid_C, PF02872; green) domains. The catalytic, metal, and substrate-binding sites are schematically indicated (see also S3 Table). The grey helix signifies the region linking the calcineurin-like phosphoesterase and 5′-nucleotidase domains of M. bovis 5′-NT. The position of mTn insertion in the 5′ nucleotidase encoding gene is shown by a black arrow. SP, signal peptide sequence. The numbers on top indicate amino acid position. (B) Phylogenetic tree construction of 5′-NTs. The rooted phylogenetic tree of the M. bovis PG45 5′-NT was generated through multiple sequence alignment and phylogenetic reconstructions. ClustalW and the "build" function of ETE3 3.1.2 by Huerta-Cepas [38] were employed for these analyses, as implemented on GenomeNet (https://www.genome.jp/tools/ete/). The tree construction utilized fast tree with slow NNI and MLACC = 3 [39]. Bootstrap values are indicated at branching points, providing confidence in the tree topology. Accession numbers follow the species’ names, and the percent identity with the 5′-NT of M. bovis PG45 is presented in parentheses. *—percent identity with the 5′-NT of A. laidlawii (total 574 aa) was calculated over a 243-aa alignment only. Bovine Mycoplasma species without identified 5′-NT available in genomes databases (April 2024) include M. alvi, M. tauri, M. testudinis, M. canadense, M. bovoculi, M. arginine, M. leachii, M. mycoides subsp. mycoides SC and M. wenyonii.
Fig 2.
Impact of 5′ nucleotidase disruption on M. bovis growth phenotype in axenic conditions.
(A) Growth curve analysis of M. bovis PG45 WT, mutants mnuA::Tn and 0690::Tn and complemented strain 0069::Tn::pOH/P_p0690 under cultivation in modified FF medium. Mycoplasma titers were determined every 24 hours over a total incubation period of 72 hours. The data are presented as the means of three independent assays, with standard deviations indicated by error bars. The hatched rectangle shows a delay in the growth rate of the 0690::Tn mutant observed during first 24 hours. (B) Statistical significance between the growth rates of the WT, the 0690::Tn and its complementation at 24-hour time point was assessed using an unpaired t-test. P values are indicated by asterisks (*P<0.05; **P<0.001). (C) Micrographs of M. bovis PG45 (WT), mnuA::Tn and 0690::Tn mutants, and the complemented 0069::Tn::pOH/P_p0690 colonies, grown on modified FF agar for 6 days. The images were captured under a light microscope using the same settings and magnification (x 2.5).
Fig 3.
In vitro characterization of the mnuA::Tn and 0690::Tn mutants.
(A) The hydrolytic activity of the 0690::Tn mutant towards adenosine-related nucleotides (ATP, ADP, 5′- and 3′-AMPs), guanosine-related nucleotides (GTP, GDP, 5′-GMP), as well as 5′-CMP, 5′-UMP and deoxynucleotides dAMP and dGMP was evaluated by assessing phosphate content (inorganic phosphate release) after incubation of mycoplasma cultures with the indicated substrates for 30 min at 37°C. The release of inorganic phosphate was measured using a malachite green phosphate colorimetric assay kit. The data represent the phosphate content (%) of the 0690::Tn mutant and its complemented 0690::Tn::pOH/P_p0690 strain relative to the WT. Results are derived from three independent experiments, each conducted in triplicate, and are presented as the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P<0.05 **P<0.01 ***P<0.001 ****P<0.0001. (B) Nuclease activity of M. bovis Triton X-114-fractionated hydrophobic protein fractions measured by Real-time PicoGreen DNase assay. The assay conditions are described in Materials and Methods. The fluorescence signal expresses amount of the dsDNA measured over 5 hours at 37°C using the SpectraMax i3 multiple detection microplate reader. The results are represented as the mean.
Fig 4.
Inactivation of MnuA and 5′-NT reduced bacterial fitness and virulence of M. bovis PG45 in mammary glands.
Mammary virulence of M. bovis PG45 and attenuation of the mnuA::Tn and 0690::Tn mutants were demonstrated in lactating BALB/c mice following intramammary challenge of L4 and R4 glands with approximately 109 CFUs of bacteria. Bacterial colonization observed after challenge with mutant strains mnuA::Tn and 0690::Tn was significantly reduced (scatter plot in panel A), with each data point representing a single gland, and the horizontal bars indicating the median of data from three or more mice. Bacterial counts of 0690::Tn mutant were rescued following co-challenge with WT M. bovis PG45 (A). Disease manifestation was characterized by neutrophil recruitment into alveolar milk spaces (black arrows) and demonstrated in representative microscopic images of H&E and anti Ly6G (neutrophil marker) immunohistochemical staining (round insets in B) of paraffin section from WT, mnuA::Tn and 0690::Tn challenged glands (B; top, middle, and bottom panels, respectively). Using RT-qPCR, the relative expression (ΔΔCt) of the neutrophil marker Ly6G (C) and inflammatory marker genes MIP2, KC, Nos2, TNFα, IL1β, and IL6 (D), was quantified relative to RNA samples extracted from the mammary tissues of normal non-challenged lactating control mice. Data are presented as box plots showing higher neutrophil recruitment and expression of inflammatory markers following challenge with WT bacteria compared to the mutant strains. Statistical significance was determined by non-parametric Mann–Whitney two-independent-samples test, with a P value of 0.05 or less considered significant. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars; 200 μm and 500 μm are shown (B).
Fig 5.
Loss of mammary fitness and virulence by M. bovis 0690::Tn mutant.
Lactating cows were treated by IMM infusion with WT, mnuA::Tn, and 0690::Tn bacterial strains or PBS as non-challenged controls. Scatter plots show daily (days 1–7 after challenge) bacterial counts (CFU/ml; A), somatic cell counts (SCC; B), and individual quarter level of clinical mastitis scores (CMS; C) for each treatment in challenged glands. Daily means and SD are shown by larger dots and error bars. Plots were constructed using SuperPlotOfData [47].
Fig 6.
M. bovis 0690::Tn mutant failed to elicit the recruitment of neutrophils into milk spaces.
H&E images of bovine mammary glands challenged with PBS (A&B), M. bovis PG45 (C&D), mnuA::Tn (E&F) and 0690::Tn (G&H) mutants. Challenge with the WT (C&D) and the mnuA::Tn mutant (E&F) elicited the recruitment of neutrophils (black arrows) into milk spaces, while immune cells populations were absent in milk spaces of mammary glands challenged with PBS (A&B) and the 0690::Tn mutant (G&H; red arrows).