Phase Transition of the Bacterium upon Invasion of a Host Cell as a Mechanism of Adaptation: a Mycoplasma gallisepticum Model

What strategies do bacteria employ for adaptation to their hosts and are these strategies different for varied hosts? To date, many studies on the interaction of the bacterium and its host have been published. However, global changes in the bacterial cell in the process of invasion and persistence, remain poorly understood. In this study, we demonstrated phase transition of the avian pathogen Mycoplasma gallisepticum upon invasion of the various types of eukaryotic cells (human, chicken, and mouse) which was stable during several passages after isolation of intracellular clones and recultivation in a culture medium. It was shown that this phase transition is manifested in changes at the proteomic, genomic and metabolomic levels. Eukaryotic cells induced similar proteome reorganization of M. gallisepticum during infection, despite different origins of the host cell lines. Proteomic changes affected a broad range of processes including metabolism, translation and oxidative stress response. We determined that the activation of glycerol utilization, overproduction of hydrogen peroxide and the upregulation of the SpxA regulatory protein occurred during intracellular infection. We propose SpxA as an important regulator for the adaptation of M. gallisepticum to an intracellular environment.

until the logarithmic growth phase. The cell pellet was obtained by centrifugation at 8,000g at 4 °C, 10 min. Samples were examined with LSM 510 Meta confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) using x63 oil objective (NA 1.40). Diode (405 nm) and heliumneon (543 nm) lasers were used for specific excitation of the fluorescent dyes DAPI and Alexa Fluor 568, respectively. The Zstacks of fluorescent images were collected at 0.370.38 µm interval.

MRM LCMS/MS analysis
To obtain transition list for proteins of interest, peak lists with protein identification (.group files) obtained for IDA experiments were loaded into Skyline software, where no more than 10 unique peptides per protein with global FDR rate from fit according to PSPEP were selected with additional constrains on peptide length (720 amino acid residues), cleavage (fully tryptic peptides without missed cleavage sites or potential ragged ends) and modifications (only carbamidomethylation of cysteins allowed). For each precursor of charge 2, 3 or 4, 3 or 4 most intense fragment ions were selected. Raw data was obtained by duplicate injection of each sample with the same chromatographic setup with total cycle time of 3.3 s. After each sample correction for retention time drift was applied, scheduled MRM time window for each precursor comprised 5 minutes.
Processing of data included peak selection (with manual review), peak integration and export utilizing Skyline software. Further analysis was performed by homemade script in R.
The algorithm included scaling normalization of LCMS replicates on peptide level and of each set of samples on protein level (based on 2 most intense peptides). Proteins with mean change across 3 biological replicates was above the threshold of 1.2 were reported as significant.

SpxA overexpression in M. gallisepticum
The transformation of M. gallisepticum was performed by electroporation as described previously 1 . Transformants were selected on a semiliquid medium supplied with 2 µg/ml of tetracyclin. The transposon insertions were confirmed and mapped by PCR and Sanger sequencing from the chromosome. The overexpression of the spxA gene was confirmed by realtime PCR.

Determination of hydrogen peroxide production
The hydrogen peroxide production in M. gallisepticum during intracellular localization was determined using the Amplex® Red Hydrogen Peroxide / Peroxidase Assay Kit (Thermo Fisher Scientific Inc, USA). 50μl of the standard curve samples, controls and suspension of eukaryotic cells and M. gallisepticum were pipeted into individual wells of a microplate. The standard curve samples were made in the mix of media for eukaryotic cells and media for M.gallisepticum. HD3 cells incubated with media for M. gallisepticum were used as a control samples. 50μl of the Amplex® Red reagent / HRP working solution was added to each microplate well and incubated for 1hour at room temperature in the dark.

Fluorescence was measured with a fluorescence microplate reader ( xMark Microplate
Spectrophotometer, BioRad ) using absorbance 560nm. Background fluorescence, determined for a noH 2 O 2 control reaction, has been subtracted from each value. Experiments were done in three independent experiments in triplicate.

Measurement of ATP concentration in M. gallisepticum cells
Cultures of M. gallisepticum control strain and MIEC were taken in latelog phase.
Normalization to the cell number was performed using quantitative RTPCR. To determine the extracellular ATP in the microcuvette were added 20 μl of sample and 100 μl ATPreagent (Lyumtek, Russia). An ATPbioluminescence integral signal was measured within 30 seconds (RLU/s) at luminometer LUM1 (Lyumtek, Russia). The measurement was carried out in three independent experiments performed in duplicate. The 7.5 * 10 9 M ATP control solution (Lyumtek, Russia) was prepared in mycoplasma culture medium. To determine the total ATP were taken 50 μl of the sample in microtube, added 450 μl of anhydrous dimethylsulfoxide and mixed thoroughly. After 1 min 20 μl of obtained extract were added to microcuvette and ATP levels were measured as described above. ATP concentration was calculated by the formula: [ATP] sample = 10•7,5•10 9 = 7,5 •10 8 ( (I sample )/ (I control )), mol/l The data represent the mean (±SD) of three independent experiments.