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

Abstract

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.

Figure 1. Invasion of M. gallisepticum into three eukaryotic cell lines under acute infection conditions.

(A) The percentage of invasion was calculated by dividing the CFU value obtained after gentamicin treatment with the CFU value of total mycoplasmas added for infection and multiplied by 100. The data represent the mean (±SD) of three independent experiments performed in triplicate. (B–D) Fluorescence was visualized with confocal microscopy. Alexa Fluor 568 phalloidin fluorescence showing eukaryotic cell F-actin stained red, DAPI fluorescence showing cell nuclei and mycoplasmas stained blue. Scale bars, 15 μm. (B) HD3 cells, (C) HeLa-229 cells, (D) mES cells. In addition, the intensity distributions of DAPI and phalloidin confirm the intracellular localization of M. gallisepticum in HD3, HeLa and mES cells, which is further illustrated by the RGB profiles seen in (E–G), respectively. The pink line in (B–D) represents the location of RGB profile analysis.

Figure 2. 2-D DIGE analysis of M. gallisepticum isolated from HD3 cells and overexpressed spxA.

(A) M. gallisepticum after 24 h of infection (Cy3, green) and laboratory strain M. gallisepticum S6 (Cy5, red). (B) M. gallisepticum after chronic infection (19 days) (Cy3, green) and laboratory strain M. gallisepticum S6 (Cy5, red). (C) M. gallisepticum after chronic infection (7weeks) (Cy3, green) and laboratory strain M. gallisepticum S6 (Cy5, red). (D) SpxA-overexpressing M. gallisepticum (Cy3, green) and laboratory strain M. gallisepticum S6 (Cy5, red). (E) Schematic construction of a transposon vector for overexpression of the spxA gene. RBS-ribosome binding site; OIR and IIR– inverted repeats.

Figure 3. Radar chart of proteins in M. gallisepticum isolated from three different cell lines after acute infection and chronic infection in HD3 cells compared with the control laboratory M. gallisepticum S6 strain using differential 2D gel electrophoresis.

M. gallisepticum initially isolated from HD3 cells–green line, M. gallisepticum initially isolated from Hela cells–violet line, M. gallisepticum initially isolated from mES cells–pink line, M. gallisepticum–orange line. The black circle represents the threshold (the ratio of the protein level in the intracellular mycoplasma relative to the control strain is (1). Values based on the ratio of the fluorescence intensity for the channels Cy3/Cy5 were counted using the PDQuest software package (Bio-Rad). MIEC marked by green Fluorescent dye (Cy3), a control laboratory strain of M. gallisepticum S6-red (Cy5). Detailed information for the proteins can be obtained in Table S1. (A) 24-h acute infection. (B) 19-days chronic infection. (C) 7-weeks chronic infection.

Figure 4. 2D DIGE analysis of various M. gallisepticum isolated from HD3 cells after 7 weeks of infection (Cy3, green) and the laboratory strain M. gallisepticum S6 (Cy5, red).

(A) M. gallisepticum (green). (B) M. gallisepticum initially isolated from HD3 cells (green). (C) M. gallisepticum initially isolated from HeLa cells (green). (D) M. gallisepticum initially isolated from mES cells (green).

Figure 5. Production of hydrogen peroxide by M. gallisepticum.

(A) Overview of pyruvate metabolism in M. gallisepticum S6. LDH-lactate dehydrogenase; PDH-pyruvate dehydrogenase complex; PTA–phosphotransacetylase; ACK-acetate kinase; NOX–NADH-oxidase. (B) Schematic illustration of the recovery pathway of glycerol and glycerol-3-phosphate in Mycoplasma gallisepticum. Free glycerol is transported into the bacterial cell by the membrane transporter GlpF. Then, the glycerol is phosphorylated by glycerol kinase GlpK, thereby forming a molecule of glycerol-3-phosphate. In addition, M. gallisepticum recycles free glycerol-3-phosphate through a transport system containing UgpACE ABC transporters. Further, glycerol-3-phosphate is converted to dihydroxyacetone phosphate by the oxidoreductase GlpO, secreting hydrogen peroxide. СМ–cell membrane; DHAP-dihydroxyacetone phosphate. (C) Determination of hydrogen peroxide release. Detection of H₂O₂ production during incubation of the eukaryotic cells HD3 with M. gallisepticum for 0, 2, 4 and 6 hours of co-cultivation using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific Inc, USA). Error bars indicate standard deviation (based on three independent experiments).

Figure 6. Distribution of SNPs localized in the VlhA cluster 4 genes in MIEC after acute (24 h) and chronic (7 weeks) infections.

Lines indicate location of SNPs detected in M. gallisepticum isolated from HD3 cells. Black line-acute infection; blue line–chronic 7-week infection; red line–both acute and chronic 7-week infection.

Figure 7. Measurement of the ATP concentration.

Detection of ATP production in MIEC after acute and chronic infections and control M. gallisepticum S6. The data represent the mean (±SD) of three independent experiments performed in duplicate.