Molecular Basis of the Slow Growth of Mycoplasma hominis on Different Energy Sources

Abstract

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis, representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria.

Keywords: Mycoplasma hominis; antibiotic sensitivity; proteomics; slow growth; thymidine.

Figure 1

The growth curves of M. hominis on medium supplemented with arginine or thymidine. (A) Growth curves of M. hominis. All points are averages of triplicate experiments with standard deviation error bars (B) M. hominis was cultured in BHI media containing phenol red with arginine or thymidine. Arginine utilization and as a consequence production of ammonia was indicated by the color change of indicator to pink. The OD560 values were measured in three time point – after dilution of the third passage culture of M. hominis H34 in appropriate medium (Init), 0 h for both culture; in the mild-log phase (Log), 30 h for arginine supplemented culture, 42 h for thymidine; and in the stationary phase (Stat), 45 h and 65 h, respectively.

Figure 2

Susceptibility of M. hominis to antibiotics. M. hominis was cultivated on an arginine or thymidine-supplemented medium with different antibiotic concentration range: 0.25, 1, 2.5, 5, 10 and 50 μg/mL. Differences between Cq-values for antibiotic treatment samples and positive control samples without treatment are shown. All bar-plots are averages of triplicate experiments with standard deviation error bars.

Figure 3

The effect of different carbon sources on M. hominis biofilm formation. Biofilm production was quantified by measuring the absorbance (560 nm) of crystal violet in a microplate. Groups are significantly different, pairwise comparisons using Wilcoxon rank sum test, p-value = 5e-09.

Figure 4

Proteins implicated in the arginine deiminase pathway and utilization of pyrimidine deoxynucleoside as energy source. Comparative proteomic analysis of M. hominis grown on thymidine-supplemented medium relative to arginine-supplemented medium. The down-regulated proteins are indicated red, up-regulated - in green, unchanged - in grey. Unknown proteins are in light grey. The full names of proteins are given in Supplementary Tables S3 , S4 .

Figure 5

Response of translation apparatus to a change in the energy source. The down-regulated proteins are indicated in red, up-regulated - in green. The corresponding full names of proteins are given in Supplementary Tables S3 , S4 .

Figure 6

Difference in the membrane-associated proteins under thymidine conditions comparing with arginine conditions. The down-regulated proteins are indicated red, up-regulated - in green, unchanged - in grey. The data about signal peptides, Lipo-box, transmembrane helices and functional domains was used in protein diagramming. The SRP-mediated protein targeting is shown. LP - lipoprotein. Only two significantly changed transmembrane proteins are not shown: PotC and PepF. The corresponding full names of proteins are given in Supplementary Tables S3 , S4 .