A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity

Abstract

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-¹³C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H₂O₂ production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

Keywords: glycerol metabolism; metabolomics; mycoplasma.

FIG 1

Overview of glucose, fructose, and glycerol uptake and metabolism by M. gallisepticum based on experimental data and genomic analyses. Question marks indicate that the transporter activity is not yet experimentally validated, but GtsA is predicted to transport glycerol, and MalF is the focus of the research described here. DHAP, dihydroxyacetone phosphate.

FIG 2

Growth of the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant in MB and MB-glycerol medium.

FIG 3

Fold change differences (log₂) in the abundance of metabolites related to glycerol metabolism in the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant in two independent untargeted metabolite profiling experiments on GC-MS (a) and LC-MS (b) platforms. Only metabolites with statistically significant differences in abundance are shown (Holm-Bonferroni corrected t test; false discovery rate cutoff, 0.05).

FIG 4

Metabolites labeled in the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant after culture in medium containing [U-¹³C]glycerol. The scale indicates the proportion of the metabolite containing the label. Values were normalized to the ¹³C/¹²C ratio in the medium. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Holm-Sidak corrected t test). A “?” indicates that the transporter is unknown (but is suggested to be GtsA based on the RT-qPCR results in this study).

FIG 5

Comparative analysis of transcripts from genes involved in glycerol transport and metabolism in the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant cultured in MB (a) or MB-glycerol (b) medium for 6 or 12 h. *, P < 0.05; **, P < 0.01 (Holm-Sidak corrected t test).

FIG 6

Hydrogen peroxide level scores for cultures of the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant (Mann-Whitney test; *, P < 0.05).

FIG 7

Transmission electron micrographs of the M. gallisepticum wild-type strain AP3AS in the presence (a) or absence (b) of glycerol and of M. gallisepticum ΔmalF mutant cells in the presence (c) or absence (d) of glycerol. Scale bar, 2 μm. Arrows indicate an electron-dense region, which is probably the terminal bleb, in some of the wild-type cells.

FIG 8

Average cell size of the M. gallisepticum wild-type strain AP3AS and the ΔmalF mutant in the presence or absence of glycerol. ***, P < 0.001; ****, P < 0.0001 (one-way analysis of variance, followed by Tukey’s multiple-comparison test).