Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling

Abstract

Background: The respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas.

Methods: In this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico.

Results: Even though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species.

Conclusions: We were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species.

Keywords: Hydrogen peroxide; Metabolic network; Metabolism; Mollicutes; Mycoplasma; Whole-genome metabolic reconstruction.

Fig. 1

Venn diagrams representing the comparison of refined networks. Numbers represent the exclusive and common reactions present in the refined networks (a) between species, and (b) between selected strains of M. hyopneumoniae. This analysis shows that most of the metabolism is common to all organisms. MHR: M. hyorhinis; MHP: M. hyopneumoniae; MFL: M. flocculare

Fig. 2

Distribution of the model reactions in the subsystems. The 457 reactions present in the model iMFpan were separated into (a) biological subsystems and (b) further into reaction types, with the exclusion of exchange reactions in this analysis

Fig. 3

Global energy and carbohydrate complete models. Metabolites are depicted in dark green and separate enzymatic activities for M. hyorhinis, M. hyopneumoniae and M. flocculare can be seen in yellow, pink and blue, respectively. Whenever an enzyme is missing from the three species, the enzyme rectangle is depicted in grey. Complete list of metabolite abbreviations and EC numbers can be found in Additional file 4

Fig. 4

Lipid, amino acid, nucleotide and cofactor complete models. Metabolites are depicted in dark green and separate enzymatic activities for M. hyorhinis, M. hyopneumoniae and M. flocculare can be seen in yellow, pink and blue, respectively. Whenever an enzyme is missing from the three species, the enzyme rectangle is depicted in grey. Complete list of metabolite abbreviations and EC numbers can be found in Additional file 4

Fig. 5

Cultivation curves in defined and complex media by species. Cell concentrations were estimated by the CCU method and error bars were calculated as the standard deviation between triplicate time-matched samples. As expected, the three species had better growth rates in the complex medium than in defined media. M. hyopneumoniae strain J (MHP); M. flocculare strain 27716 (MFL); M. hyorhinis strain ATCC17981 (MHR)

Fig. 6

Distinct products of the metabolism of pyruvate from growth in complex Friis medium and Yus+ medium of M. hyopneumoniae strains 7448 (MHP_7448) and J (MHP_J), M. flocculare strain 27716 (MFL_27716) and M. hyorhinis strain ATCC17981 (MHR_17981). In complex medium, we calculated the ratio between the peak signal in cultivated versus control medium and error bars were calculated as the standard deviation between triplicate time-matched samples. For defined medium, we detected the actual concentration for the metabolites and error bars were calculated as the standard deviation between duplicate time-matched samples. a In complex medium, M. hyopneumoniae (both strains) and M. flocculare can produce high amounts of acetate; the yields are even higher from M. hyopneumoniae. M. hyorhinis, on the other hand, produces low concentrations of acetate in this medium. The final glycolysis product for M. hyorhinis is thus pyruvate. b In defined medium, M. hyopneumoniae (strain J) and M. flocculare produce similar amounts of acetate while M. hyorhinis contains only residual levels of this metabolite. The three species can produce low amounts of formate in both media

Fig. 7

Differential metabolism of M. hyorhinis, M. hyopneumoniae and M. flocculare. Metabolites are depicted in dark green and separate enzymatic activities for M. hyorhinis, M. hyopneumoniae and M. flocculare can be seen in yellow, pink and blue, respectively. Whenever an enzyme is missing from the three species, the enzyme rectangle is depicted in grey. Complete list of metabolite abbreviations and EC numbers can be found in Additional file 4