RESUMEN
Introduction. Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.
Asunto(s)
Mycoplasma hominis , Proteoma , Timidina/metabolismo , Arginina , Humanos , Infecciones por Mycoplasma , Mycoplasma hominis/genética , Mycoplasma hominis/metabolismo , Fenotipo , Fosfatos , RibonucleósidosRESUMEN
Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9â¯h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.