Comparative Metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum Reveals Fundamental Differences in Active Metabolic Pathways and Suggests Novel Gene Annotations

Abstract

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.

SdrA, an NADP(H)‐regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly

“…Following centrifugation, the clear upper aqueous phase was collected into a fresh microcentrifuge tube and dried in rotational vacuum concentrator (RVC‐2‐33; John Morris Scientific) with 30 μl of methanol added to the final drying step. The pulled point inserts were sealed in GC/LC vials and derivatised using methoxyamine (Sigma) and N,O‐bistrimethylsilyltrifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS; Thermo Scientific) before analysis of polar metabolites using the Shimadzu GC‐QQQ as described previously (Best et al, ; Masukagami et al, ).…”

“…Molecular masses of authentic standards from an in‐house Metabolomics Australia library and retention times were used for metabolite identification. Log‐transformed and median‐normalised data were statistically analysed by the online tool MetaboAnalyst (http://www.metaboanalyst.ca/) and R‐script using an R‐based statistical analysis package from Metabolomics Australia, as described previously (Masukagami et al, ). The level of abundance of metabolites between C. burnetii NMII versus the sdrA mutant and complemented mutant versus the sdrA mutant were compared using unpaired Student's t test with the BH adjustment to correct for false discoveries, with p < .05 considered significant.…”

“…To induce phase separation, the supernatant was adjusted to entific) before analysis of polar metabolites using the Shimadzu GC-QQQ as described previously (Best et al, 2018;Masukagami et al, 2017).…”

“…Molecular masses of authentic standards from an in-house Metabolomics Australia library and retention times were used for metabolite identification. Logtransformed and median-normalised data were statistically analysed by the online tool MetaboAnalyst (http://www.metaboanalyst.ca/) and R-script using an R-based statistical analysis package from Metabolomics Australia, as described previously (Masukagami et al, 2017).…”

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…M. bovis does not ferment glucose or hydrolyse arginine (Khan et al., ), but instead uses organic acids such as lactate and pyruvate, as energy sources for growth. A recent metabolomics study has greatly increased both our knowledge base of key metabolites and our understanding of potential pathways important for M. bovis growth in vitro (Masukagami et al., ). One product of metabolism is hydrogen peroxide, which may be a pathogenicity factor in mycoplasma infections (Khan, Miles, & Nicholas, ; Zhao et al., ).…”